ان الكتاب أن نعترف مالكوم كامبل وتود Eckdahl مع اتحاد الجينوم لتعليم نشط (GCAT) والتحالف التعليم HHMI-العلوم - الصيادون فج النهوض علم الجينوم وعلم التطوري برنامج (SEA-فاجات). وقد أيد هذا المشروع من المنح المقدمة من المركز الوطني لبحوث الموارد (P20RR016460) والمعهد الوطني للعلوم الطبية العامة (P20GM103429) من المعاهد الوطنية للصحة. وأيد هذا البحث في جزء من مؤسسة العلوم الوطنية في إطار منحة # IIA-1457888. وقدمت المؤسسية بالإضافة إلى (جامعة أواتشيتا المعمدان) الأموال من خلال دينار باترسون الصيف زمالة أبحاث.

1. تصميم وترتيب أليغنوكليوتيد] مع ينتهي مثبت المناسبة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحديد الإنهاء مستقلة رو المحتملة من خلال التحليل الجيني باستخدام البرامج التي هي متاحة بحرية على الانترنت. 7 </li><li style=";text-align:right;direction:rtl"> عند العمل مع الحمض النووي المزدوج تقطعت بهم السبل، وتحديد اتجاه فاصل لفحصها. 7 البلازميد الموارد الوراثية النباتية-الأزرق يتحقق إلا الإنهاء ligated في 5 &#39;إلى 3&#39; الاتجاه على رأس (حبلا إلى الأمام). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحويل حبلا فاصل أسفل (عكس) لتكملة لها عكس لإعادة توجيه تسلسل للاختبار في مكتب المدعي العام الاتحادي الأزرق باستخدام البرمجيات الحرة على الانترنت، على سبيل المثال، القرد. </li></ol></li><li style=";text-align:right;direction:rtl"> بعد تحديد التوجه أن يكون صحيحا، إضافة نهاية لزجة &quot;5&#39;-CGAC-3&quot; إلى 5 &#39;نهاية حبلا العلوي. إضافة نهاية لزجة &quot;5&#39;-CCGC-3&quot; إلى 5 &#39;نهاية حبلا السفلي. 8 وهذا سيضمن سيتم إدراج أن إدراج ligated في المناسبةالتوجه باستخدام GGA. </li><li style=";text-align:right;direction:rtl"> مرة واحدة يتم تحديد تسلسل، نظام كما أليغنوكليوتيد] الذين تقطعت بهم السبل واحدة تجاريا. </li></ol> 2. التليين أليغنوكليوتيد] (ينطبق فقط لتجميد المجفف DNA) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اعادة تعليق [أليغنوكليوتيد الفردية في المياه الحرة نوكلياز إلى تركيز 100 ميكرومتر. </li><li style=";text-align:right;direction:rtl"> جعل العازلة 10X الصلب: 1 M كلوريد الصوديوم و 100 ملي تريس، حمض الهيدروكلوريك 7.4 درجة الحموضة. ويمكن تخزين عازلة الصلب في 4 درجات مئوية لعدة أسابيع. مرة واحدة يتم عازلة الصلب وإعادة تعليق أليغنوكليوتيد]، يبدأ الصلب 8. </li><li style=";text-align:right;direction:rtl"> لكل فاصل لفحصها، إضافة 16 ميكرولتر من الماء نقي للغاية لأنبوب microcentrifuge 1.5 مل. </li><li style=";text-align:right;direction:rtl"> إضافة 2 ميكرولتر من العازلة 10X الصلب إلى كل أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من كبار النوكليوتيد حبلا (100 ميكرومتر) من فاصل المطلوب. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من النوكليوتيد أسفل حبلا (100 ميكرومتر) من فاصل المطلوب. </li><li style=";text-align:right;direction:rtl"> إنشاء الغليان با المياهعشر باستخدام الدورق 1000 مل تحتوي على ما يقرب من 400 مل من ماء الصنبور ومكان على طبق ساخن. </li><li style=";text-align:right;direction:rtl"> وضع أنابيب microcentrifuge من الخطوة 2.6 في تعويم وتغلى لمدة 4 دقائق. بعد 4 دقائق، إيقاف لوحة الحرارة ولكن ترك الأنبوب وتطفو في حمام مائي لببطء بارد O / N (18 ساعة) 8. تخزين الإنهاء صلب عند درجة حرارة -20 درجة مئوية. </li></ol> 3. الذهبي الجمعية بوابة (ستاندرد ميكس - التكلفة الفعالة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل 40 نانومتر التخفيف من الإنهاء صلب. إضافة 124 ميكرولتر من المياه nuclease مجانا إلى 1 ميكرولتر من مزيج الإنهاء صلب (100 ميكرومتر). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات الطرد المركزي لمدة 30 ثانية في 10000 x ج ومتجر على الجليد. </li></ol></li><li style=";text-align:right;direction:rtl"> الى ذلك مناسبا أنبوب جديد لthermocycler المستخدمة، إضافة 6 ميكرولتر من المياه nuclease مجانا. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من الحمض النووي التجاري يغاز العازلة [300 ملي تريس، حمض الهيدروكلوريك (الرقم الهيدروجيني 7،7-7،8)، و 100 ملي MgCl 2، 100 ملي DTT و 10 ملي اعبي التنس المحترفين]. تنفيذ جميع الخطوات اللاحقة على الجليد. </li><li style=";text-align:right;direction:rtl"> إضافة1 ميكرولتر من الموارد الوراثية النباتية الأزرق (35-50 نانوغرام / ميكرولتر) بلازميد الوجهة إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من 40 نانومتر الإنهاء ملدن إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 0.5 ميكرولتر التجاري عالية الدقة BsaI تقييد نوكلياز داخلية للأنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 0.5 ميكرولتر من يغاز الحمض النووي التجاري إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي خليط التفاعل في 10000 x ج لمدة 120 ثانية. ثم وضع أنبوب مباشرة في thermocycler. </li><li style=";text-align:right;direction:rtl"> تشغيل برنامج thermocycler: 20 دورات من 1 دقيقة عند 37 درجة مئوية تليها 1 دقيقة في 16 درجة مئوية، تليها 1 حلقة 15 دقيقة عند 37 درجة مئوية. استخدام عينات مباشرة أو تجميد وتخزين في درجة حرارة -20 درجة مئوية. 8 </li></ol> 4. الذهبي الجمعية بوابة (التجاري ميكس ماجستير - الوقت الفعال) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 124 ميكرولتر من المياه nuclease مجانا إلى 1 ميكرولتر من 100 ميكرومتر صلب الإنهاء بإنشائه في الخطوة 2. وهذا يخفف من تركيز الحمض النووي فاصل إلى 40 نانومتر.تنفيذ هذه الخطوة بشكل جديد، وصفت 1.5 مل أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> الى ذلك مناسبا أنبوب جديد لthermocycler، إضافة 14 ميكرولتر من المياه nuclease مجانا. تأكد من وصفت أنبوب جديد وفقا لذلك. </li><li style=";text-align:right;direction:rtl"> إضافة 2 ميكرولتر من التجاري ماستر العازلة مزيج إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من الموارد الوراثية النباتية-الأزرق البلازميد (35-50 نانوغرام / ميكرولتر) إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 2 ميكرولتر من 40 نانومتر الإنهاء ملدن إلى أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من التجاري ميكس ماجستير في أنبوب microcentrifuge. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي أنبوب نانومتر 40 في 10000 x ج لمدة 120 ثانية. ثم وضع أنبوب مباشرة في thermocycler. </li><li style=";text-align:right;direction:rtl"> تشغيل برنامج thermocycler: 1 حلقة 60 دقيقة عند 37 درجة مئوية تليها 1 حلقة 15 دقيقة في درجة حرارة 55 مئوية. استخدام عينات مباشرة أو تجميد وتخزين في درجة حرارة -20 درجة مئوية. </li></ol> 5. التحول واختيار مستعمرة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد قاعدة لوريا (LB) أجار لوحات بتري تحتوي على 1 ملم ويركزأيون من الأمبيسلين (الأمبير) وتركيز 10 ملم من أرابينوز (ARB). إعداد لا يقل عن 10 مل من 1 M L حل الأسهم -arabinose لأن هذا الحل الأسهم ستستخدم في خطوات لاحقة. </li><li style=";text-align:right;direction:rtl"> استخدام أي عالية الكفاءة (10 أغسطس - 10 سبتمبر transformants / ميكروغرام) المختصة كيميائيا E. القولونية (JM109) خلايا للتحول. 9 للحصول على أفضل النتائج، إضافة 3-5 ميكرولتر من رد فعل ربط إلى 50 ميكرولتر من الخلايا المختصة. عند استخدام الخلايا المختصة الكيميائية، اتبع بروتوكول التحول محددة للخلايا المستخدمة. </li><li style=";text-align:right;direction:rtl"> نشر نصف (25 ميكرولتر) من الخلايا تحولت إلى LB قبل تحسنت (أمبير / ARB) أجار لوحات باستخدام على شكل حرف L العقيمة &quot;عصا الهوكي&quot; واحتضان O / N عند 37 درجة مئوية. نظرا لمعدل الأمبيسلين السريع من التحلل، لا احتضان عند 37 درجة مئوية خلال 24 ساعة. </li><li style=";text-align:right;direction:rtl"> أداء اختيار مستعمرة على أساس اللون عن طريق التفتيش البصري. وينبغي أن يكون الربط الناجح أبيض / أصفر في الضوء المرئي ويتألق الأخضر undeص الأزرق (450 نانومتر) أو الأشعة فوق البنفسجية. ومن شأن ربط غير ناجحة تنتج مستعمرة هذا هو الازرق في اللون تحت الضوء المرئي بعد 18-20 ساعة. </li></ol> 6. التحقق والكمي لالمنهي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 5 مل أنابيب معقمة LB-مرق مع 1 ملم الأمبيسلين و 10 ملي أرابينوز. عدد الأنابيب اللازمة يعتمد على عدد من الإنهاء التي يجري اختبارها بالإضافة إلى ضوابط (خلايا مقاومة الأمبيسلين التي لا يتألق والخلايا التي تحتوي على البلازميد الموارد الوراثية النباتية-الأزرق غير مختصر). </li><li style=";text-align:right;direction:rtl"> تحت الضوء المرئي اختيار 2-3 / مستعمرات صفراء البيضاء (الخطوة 5.4) باستخدام حلقة عقيمة. السماح للعينات لاحتضان في المرق على شاكر عند 37 درجة مئوية و 160 دورة في الدقيقة ل16-18 ساعة. لا احتضان هذه العينات لأكثر من 24 ساعة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اختياريا، واستخدام مطياف لتحديد كثافة الخلايا الضوئية في 600 نانومتر (OD 600) لضمان نمو الخلايا كاف. عموما، فهناك علاقة OD 600 من ~ 0،8-1،0 بعد 16-18 ساعة من النمو. </li></ol></li><li style=";text-align:right;direction:rtl"> استعمالمعقمة لوحة 96-جيدا لالخطوات المتبقية. إجراء هذه الخطوات في بيئة معقمة وفي ثلاث نسخ. إضافة 199 ميكرولتر من معقم LB + أمبير / الارابينوز مرق (1 ملم الأمبيسلين / 10 ملي الارابينوز) إلى كل بئر. تشمل الغرفة على لوحة لمدة ثلاثة عناصر تحكم منفصلة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام الضوابط الثلاثة التالية: بئر (1) التي تحتوي على LB + أمبير / الارابينوز مرق فقط وكذلك (2) للخلايا المختصة هو دون تغيير (فارغ) لتكون بمثابة فارغة. لالكمي (3) من قوة فاصل، ينبغي إدراج الخلايا التي تحتوي على الموارد الوراثية النباتية الأصلية تقطيعه أو التي تحتوي على الموارد الوراثية النباتية الأزرق ligated مع غير إنهاء تسلسل (مراقبة إشارة لتقدير). انظر الشكل رقم 1 لتخطيط لوحة القارئ. </li></ol></li><li style=";text-align:right;direction:rtl"> الماصة 1 ميكرولتر من محلول الخلية من O / N المرق في الآبار الفردية من لوحة 96-جيدا. </li><li style=";text-align:right;direction:rtl"> لوحات ختم مع غطاء للتنفس ويهز ل18-20 ساعة عند 37 درجة مئوية و 160 دورة في الدقيقة. </li><li style=";text-align:right;direction:rtl"> باستخدام قارئ لوحة، وتحديد OD 600 من خلال قراءةالامتصاصية في 600 نانومتر. قياس GFP مضان كما يلي: الإثارة 395 و 509. الانبعاثات قياس RFP مضان كما يلي: الإثارة 575 والانبعاثات 605. بعد 18-20 ساعة، ينبغي أن OD 600 أن ما يقرب من 0.5. </li><li style=";text-align:right;direction:rtl"> لتطبيع للالتباين في النمو، واستخدام المعادلة: <img alt="المعادلة 1" src="/files/ftp_upload/54064/54064eq1.jpg" /> لكلا GFP وطلب تقديم العروض. </li><li style=";text-align:right;direction:rtl"> بعد التطبيع، وتحديد قوة فاصل النسبية باستخدام المعادلة <img alt="المعادلة 2" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

<ol>
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The authors have nothing to disclose.

أهم خطوة في هذا البروتوكول هو الصحيح تصميم النوكليوتيد قبل الطلب. يجب أن يكون أليغنوكليوتيد] نهايات لزجة المناسبة إضافة إلى نهايات 5 &#39;من كل من أعلى وأسفل مسارات لضمان GGA التأسيس هو ممكن. بالإضافة إلى ذلك، من المهم أن تبديل اتجاه اليسار الإنهاء التي تواجه (الإنهاء الت...

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can be completed in a relatively short time frame. 
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced. 
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pGR-Blue Plasmid</td>
			<td style="width:243px;">Addgene</td>
			<td style="width:115px;">68374</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGR-Plasmid</td>
			<td>Addgene</td>
			<td style="width:115px;">46002</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AeraSeal-(Sterile Sheets)</td>
			<td>Excel Scientific</td>
			<td>BS-25</td>
			<td style="width:235px;">Sterile Sheets only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X T4 DNA ligase Buffer</td>
			<td>NEB</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BsaI-HF</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td style="width:235px;">The non-HF enzyme will work but is less heat stable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NEB Golden Gate Assembly Mix</td>
			<td>NEB</td>
			<td>E1600S</td>
			<td style="width:235px;">Commerial Master Mix refered to in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Microcentrifuge Floating Rack</td>
			<td>Nova Tech International</td>
			<td>F18875-6401</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A-3256</td>
			<td style="width:235px;">D-Arabinose will not induce the pBAD promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Broth, Miller</td>
			<td>Sigma Aldrich</td>
			<td>L1900</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Agar , Miller&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L2025</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan-Infinite M200 Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mix &#38; Go Competent Cells - Strain JM109</td>
			<td>Zymo Research</td>
			<td>T3005</td>
			<td style="width:235px;">Use company recommended transformation protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ApE: A plasmid editor-software</td>
			<td style="width:243px;">http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl, Molecular Grade</td>
			<td>Promega</td>
			<td>H5121</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Sodium Chloride (Crystalline/Biological, Certified)</td>
			<td>Fisher Chemical</td>
			<td>S671</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comercial Oligonucleotide synthesis</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td style="width:235px;">http://www.idtdna.com/site</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Microtest Tissue Culture Plates- 96 well (Sterile)</td>
			<td>Falcon</td>
			<td>35-3072</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mycobacteriophage &#34;Bernal13&#34;</td>
			<td>Genebank</td>
			<td>KJ510413</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>IDT004</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, L-shaped Hockey-Stick Cell</td>
			<td>Life Science Products</td>
			<td>6444-S1</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nano-Drop 2000c UV-Vis Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>2000c</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td>
			<td style="width:243px;">http://rna.igmors.u-psud.fr/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

وهذا البروتوكول تنتج الخلايا التي تحتوي على الموارد الوراثية النباتية الأزرق مع فاصل ligated بين GFP وطلب تقديم العروض باستخدام الذهبي الجمعية بوابة (الشكل 2). المستعمرات الإيجابية التي تحتوي على إدراج ligated يمكن اختيار على أساس اللون. في الضوء ا...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

سرعة التحقق من صحة ترمينتورس استخدام الموارد الوراثية النباتية الأزرق البلازميد والجمعية جولدن جيت

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.6K Views.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

Video: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

تنقية DNA البلازميد من المستعمرات البكتيرية باستخدام Miniprep Qiagen كيت

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

التحول من الحمض النووي البلازميد في كولاي استخدام أسلوب الصدمة الحرارية

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

في المختبر عن طريق الجمعية النووية مجزأ مقتطفات البيض القيطم

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

التجمع ، والتحميل ، والمحاذاة لخلية نموذج تحليلي نابذة فائقة السرعة

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

الابتدائية الثقافة وElectroporation البلازميد الجهاز الفئران كورتي.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

PCR Thermocycling السريع باستخدام الحراري الحرارية الميكروسكيل

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

شاشة عالية الإنتاجية الخميرة Overexpression البلازميد

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

جيل من البلازميد ناقلات معربا عن بروتينات الموسومة FLAG تحت تنظيم الاستطالة الإنسان عامل-1α المروج عن طريق الجمعية جيبسون

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

عزل الترشيح من الأحماض النووية: وبسيطة وسريعة استخراج الحمض النووي الطريقة

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

كابريسي: الجمعية الكيميرا بواسطة الانتعاش البلازميد والقيود إنزيم إدراج الموقع

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...