無限制酶克隆(此為RF-Cloning討論版之文章備份)

Dear all,

I am using RF-Cloning for about 3 years and really enjoy the results.
Back to the time I first test the RF-Cloning protocol, it was really frustrating to get no colonies on plates.
But now I have some tips myself to increase the colony numbers and possibility of correct clones.
I hope this would help people who try RF-Cloning for the first time.

Procedure and tips:
1. Design the primers with the RF-Cloning website, try to get the Plasmid-annealing-section of primer would annealing above 59°C. Get the extension time of 2nd PCR!
2. Order the long primers that above 40 nt in PAGE quality.
3. 1st PCR should test in different buffers, take 50 ul Phusion reaction as example:

[HF buffer]
[HF buffer+MgCl2(50 mM) 1.5 ul]
[HF buffer+MgCl2(50 mM) 1.5 ul+DMSO 1 ul]
[GC buffer]
[GC buffer+MgCl2(50 mM) 1.5 ul]
[GC buffer+MgCl2(50 mM) 1.5 ul+DMSO 1 ul]

4. Gel purification of the 1st PCR target product (I use GeneJET Gel Extration and DNA Cleanup Micro Kit, Thermo Scientific), the DNA concentrations should be above 50 ng/ul, if not, repeat 1st PCR and Gel purification again.
5. Extract plasmids with Minprep kits.
6. Calculate gel-purified 1st PCR product and the plasmid as insert and vector on any ligation calculator by 3:1 ratio (insert:vector) and make the mass of insert+vector between 50 to 500 ng to increase circular form of DNA for transformation.
7. Prepare 2nd PCR as:

5x GC buffer 10 ul
MgCl2(50 mM) 1.5 ul
dNTP(2.5 mM) 4 ul
gel-purified 1st PCR product X ul (as calculation in Step 6)
plasmid X ul (as calculation in Step 6, or double the amount to get more colonies)
ddH2O 30 ul (I never change the ddH2O volume, no matter the volumes of gel-purified 1st PCR product and plasmid)
Phusion 0.25 ul

8. Run 2nd PCR procedure as:

98oC 98oC 59oC 72oC 72oC
30sec 10sec 2min time from Step1 5min
|______________x15______________|

(Yes, I found longer annealing time would get more colonies)

9. After finished 2nd PCR, immediately add 0.33 ul DpnI, mix and spin down.
10. The finial-touch cycle, run thermocycler as:

37oC 80oC 98oC 98oC 64oC 72oC
60min 20min 30sec 10sec 2min 5min

98oC 60oC 72oC 98oC 58oC 72oC
10sec 2min 5min 10sec 2min 5min

72oC 12oC
5min storage

(I do not test other conditions, since this works fine to me every time!)

11. Transformation to high efficiency competent E. coli according to [Gene. 1990 Nov 30;96(1):23-8. doi: 10.1016/0378-1119(90)90336-p.]
12. Happy colony-PCR screening~