Dandekar & Chandler: PCR "sewing"

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 27: Line 27:
'''You will need the following ingredients for the Sewing PCR mix:'''  
'''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!
+
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 #1 5.0ul  
-
 
+
:* Fragment #2 5.0ul  
-
- Fragment #2 5.0ul  
+
:* dNTPs 1.0ul  
-
 
+
:* DMSO (optional) 1.0ul  
-
- dNTPs 1.0ul  
+
:* 5x Phusion buffer 10.0ul  
-
 
+
:* ddH2O 27.5ul (or enough to 50ul total)  
-
- DMSO (optional) 1.0ul  
+
:* Phusion polymerase 0.5ul
-
 
+
-
- 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.
''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.
Line 48: Line 42:
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
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'''
'''Part I'''

Revision as of 19:02, 4 March 2014

Home        Contact        Internal        Protocols        Lab Members        Publications        Research        Talks       


Back to Protocols

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

Personal tools