[This protocol can be used for substituting individual base pairs, adding tags, making deletions, pretty much anything]
(Kyle Gurley version 1.0; 01/27/04)
Adapted from: PCR Primer -- A laboratory manual
Cold Spring Harbor Press 1995, pages 603-611
There are 3 PCR reactions The products from PCR1 and PCR2 are mixed together to become a template for PCR 3.
You need 4 primers (A, B, C, D) A and D flank your gene at the 5' and 3' end (As usual, it's best if primers A and D have restriction sites engineered into them for ease in cloning at the end). B and C are engineered to incorporate your change of interest.
For the example, lets say our gene is 1000 bp long, and I wanted to add a TAG at base pair # 200.
Next, we would mix these products in the same tube and they become a template for the last PCR. HOW ?
(The RED and BLUE we engineered into the B and C primers are complimentary to each other and they code for the tag).
For PCR 3, the first thing that happens is that we boil the tube right ?
So, upon reannealing in PCR 3, you get this:
For all 3 PCR steps, use Pfu Ultra. The stuff is great and has a very low mutation rate
(In fact, I've built about 12-15 constructs with this strategy and have only found one coding mutation).
For PCR 1 and PCR 2, use touchdown PCR:
CYCLE 30 times:
68 45'' [-0.5 degrees per cycle]
68 1'30'' (I think Pfu is roughly 1 minute per kb, but you should double check)
For PCR 3
CYCLE 24 times:
68 2' (I think Pfu is roughly 1 minute per kb, but you should double check)
1) The overhangs in primers B and C need to overlap each other by about 20 bp so that they can anneal in PCR 3 to form the template. I tried it once with 10 bp overlap and it did NOT work.
2) For all PCRs:
10X buffer 5
20mM dNTP 0.63
20uM forward primer 1
20uM reverse primer 1
Pfu ULTRA 1
TOTAL 49 uL
Add 1uL of template (for PCR 1 and PCR 2, try to use roughly between 1 and 10 ng of template)
3) For PCR 3 :
For template for PCR 3, first run a quick gel of PCR 1 and PCR 2 to make sure the reaction worked and the bands are the expected size.
(You don't need to purify the reaction products from PCR 1 and PCR 2 before PCR 3. I've tried doing that versus using raw material and both ways worked great).
Dilute the reaction products 1uL from PCR 1 + 1 uL from PCR 2 + 10uL of water and mix.
Now, use 1 ul of this mix for template for PCR 3.
4)If your goal is to make a small change, like single base pair mutation:
This protocol should work just fine. Just make your primers B and C with the base pair change incorporated into the overhang. I would make sure the change is not at the end of the overhang, but it should define the overhang. Meaning, it is the only basepair that doesn't match template, so it begins the 20bp overhang. That way, it is surrounded by proper matches on either end. Just make sure that B and C have overlap so the reaction products from PCR 1 and PCR 2 will overlap to make template later.
GOOD LUCK !!!
For PCR 3, you use primers A and D and for template, you just use the reaction products from PCR 1 and PCR 2.