IGEM:UC Berkeley/2006/OverlapExtensionRxn - Revision history

JCAnderson at 22:12, 15 November 2006

JCAnderson — Wed, 15 Nov 2006 22:12:45 GMT

←Older revision		Revision as of 22:12, 15 November 2006
Line 17:		Line 17:
	step 5: hold at 4 degC forever		step 5: hold at 4 degC forever
	'''Note that the wobble program is basically a short PCR program without extra denaturation steps'''		'''Note that the wobble program is basically a short PCR program without extra denaturation steps'''
-	#There is nothing to see on a gel, so don't bother, just go into the [[IGEM:UC_Berkeley/2006/Short_DNA_cleanup]] procedure.	+	#There is nothing to see on a gel, so don't bother, just go into the [[IGEM:UC_Berkeley/2006/Short_DNA_cleanup \| short DNA cleanup]] procedure.

JCAnderson at 22:12, 15 November 2006

JCAnderson — Wed, 15 Nov 2006 22:12:13 GMT

←Older revision		Revision as of 22:12, 15 November 2006
Line 17:		Line 17:
	step 5: hold at 4 degC forever		step 5: hold at 4 degC forever
	'''Note that the wobble program is basically a short PCR program without extra denaturation steps'''		'''Note that the wobble program is basically a short PCR program without extra denaturation steps'''
-	#There is nothing to see on a gel, so don't bother, just go into the [[Short_DNA_cleanup]] procedure.	+	#There is nothing to see on a gel, so don't bother, just go into the [[IGEM:UC_Berkeley/2006/Short_DNA_cleanup]] procedure.

JCAnderson at 22:11, 15 November 2006

JCAnderson — Wed, 15 Nov 2006 22:11:39 GMT

New page

'''Overlap extension reactions take two oligos with complementary 3' ends, and fill in the rest of the sequence with a polymerase.<br>
Any polymerase system will work for this (Taq, Vent, Expand, Pfu...)'''

#Make 100uM stocks of the oligos (the usual concentration for stocks, it's used here as 1x)
#Set up per extension reaction (it's ok to make a master mix of everything except the oligos, then split it and add the oligos--just make sure to add the polymerase after the buffer and dNTPs have been added)
34 uL ddH2O
5 uL 2mM in each dNTPs
5 uL 10x ThermoPol buffer
5 uL 100 uM oligo 1
5 uL 100 uM oligo 2
1 uL Vent polymerase
#Run the "wobble" program, which is:
step 1: 2 min at 94
step 2: 30 sec at 55 degC
step 3: 30 sec at 72 degC
step 4: Got to 2 10 times
step 5: hold at 4 degC forever
'''Note that the wobble program is basically a short PCR program without extra denaturation steps'''
#There is nothing to see on a gel, so don't bother, just go into the [[Short_DNA_cleanup]] procedure.