IGEM:UC Berkeley/2006/OverlapExtensionRxn
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< IGEM:UC Berkeley/2006(Difference between revisions)
Current revision (18:12, 15 November 2006) (view source) m |
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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. |
Current revision
Overlap extension reactions take two oligos with complementary 3' ends, and fill in the rest of the sequence with a polymerase.
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.
