Dandekar & Chandler: PCR "sewing"

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== PCR "sewing" of two fragments with overlapping regions of homology ==  
+
= PCR "sewing" of two fragments with overlapping regions of homology =
* "Sewing" is a bastardization of "SOE" which stands for Splicing by overlap/overhang extension
* "Sewing" is a bastardization of "SOE" which stands for Splicing by overlap/overhang extension
* This protocol has not yet failed Nicole even once
* This protocol has not yet failed Nicole even once
* This protocol is lifted from [http://2013.igem.org/wiki/images/2/24/PCR_Sewing.pdf| iGEM]
* This protocol is lifted from [http://2013.igem.org/wiki/images/2/24/PCR_Sewing.pdf| iGEM]
* Watson gives this protocol two paws up
* Watson gives this protocol two paws up
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[[Image:WatsonSOE2.jpg]]
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[[Image:WatsonSOE2.jpg|100 px]]
 +
 
 +
==iGEM PCR Sewing Protocol ==
 +
===Reaction Set Up===
 +
'''You will need the following ingredients for the Sewing PCR mix:'''
 +
 
 +
* If you are going to be cloning with this fragment (and you know you are) use [https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase| Phusion] for best results!
 +
- Fragment #1 5.0ul
 +
 
 +
- Fragment #2 5.0ul
 +
 
 +
- dNTPs 1.0ul
 +
 
 +
- DMSO (optional) 1.0ul
 +
 
 +
- 5x Phusion buffer 10.0ul
 +
 
 +
- ddH2O 27.5ul (or enough to 50ul total)
 +
 
 +
- Phusion polymerase 0.5ul
 +
 
 +
''Pro Tip'' - If you are doing multiple PCR’s, prepare a Master Mix by adding the common ingredients for all reactions to one pot. Add the polymerase LAST.
 +
 
 +
===Cycling Protocol===
 +
The PCR sewing reaction takes place in two parts
 +
 
 +
In the first part, you will run the usual PCR cycle for only 5 cycles in order to let the fragments prime each other and synthesize a small amount of full length DNA product
 +
 
 +
'''Part I'''
 +
 
 +
Step 1 (Denaturation): 98˚C 30 sec ''(for high GC, we use 3 min; the point here is get single strands and to linearize so that the overlapping regions can find each other)''
 +
 
 +
Step 2 (Denaturation loop): 98˚C 15 sec
 +
 
 +
Step 3 (Annealing): 50˚C – 60˚C 25 sec
 +
 
 +
Step 4 (Elongation): 72˚C 30 sec/kb
 +
 
 +
-- ''repeat steps 2-4 for a total of 5 cycles'' --
 +
 
 +
Step 5 (Final Elongation): 72˚C 5 min
 +
 
 +
Step 6 (Storage): 4˚C forever
 +
 
 +
 
 +
'''Part II'''
 +
 
 +
'''After the first 5 cycles are over, add 1.0ul of 10uM end primers to each tube'''
 +
 
 +
'''Also add 0.5ul of Phusion polymerase'''
 +
 
 +
Run the PCR for an additional 20-25 cycles, as usual
 +
 
 +
 
 +
Step 1 (Denaturation): 98˚C 30 sec
 +
 
 +
Step 2 (Denaturation loop): 98˚C 15 sec
 +
 
 +
Step 3 (Annealing): 50˚C – 60˚C 25 sec
 +
 
 +
Step 4 (Elongation): 72˚C 30 sec/kb
 +
 
 +
Step 5 (Final Elongation): 72˚C 5 min
 +
 
 +
Step 6 (Storage): 4˚C forever

Revision as of 17:59, 4 March 2014

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Contents

PCR "sewing" of two fragments with overlapping regions of homology

  • "Sewing" is a bastardization of "SOE" which stands for Splicing by overlap/overhang extension
  • This protocol has not yet failed Nicole even once
  • This protocol is lifted from iGEM
  • Watson gives this protocol two paws up

iGEM PCR Sewing Protocol

Reaction Set Up

You will need the following ingredients for the Sewing PCR mix:

  • If you are going to be cloning with this fragment (and you know you are) use Phusion for best results!

- Fragment #1 5.0ul

- Fragment #2 5.0ul

- dNTPs 1.0ul

- DMSO (optional) 1.0ul

- 5x Phusion buffer 10.0ul

- ddH2O 27.5ul (or enough to 50ul total)

- Phusion polymerase 0.5ul

Pro Tip - If you are doing multiple PCR’s, prepare a Master Mix by adding the common ingredients for all reactions to one pot. Add the polymerase LAST.

Cycling Protocol

The PCR sewing reaction takes place in two parts

In the first part, you will run the usual PCR cycle for only 5 cycles in order to let the fragments prime each other and synthesize a small amount of full length DNA product

Part I

Step 1 (Denaturation): 98˚C 30 sec (for high GC, we use 3 min; the point here is get single strands and to linearize so that the overlapping regions can find each other)

Step 2 (Denaturation loop): 98˚C 15 sec

Step 3 (Annealing): 50˚C – 60˚C 25 sec

Step 4 (Elongation): 72˚C 30 sec/kb

-- repeat steps 2-4 for a total of 5 cycles --

Step 5 (Final Elongation): 72˚C 5 min

Step 6 (Storage): 4˚C forever


Part II

After the first 5 cycles are over, add 1.0ul of 10uM end primers to each tube

Also add 0.5ul of Phusion polymerase

Run the PCR for an additional 20-25 cycles, as usual


Step 1 (Denaturation): 98˚C 30 sec

Step 2 (Denaturation loop): 98˚C 15 sec

Step 3 (Annealing): 50˚C – 60˚C 25 sec

Step 4 (Elongation): 72˚C 30 sec/kb

Step 5 (Final Elongation): 72˚C 5 min

Step 6 (Storage): 4˚C forever

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