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#1 2022-01-18 08:31:24

sekissme
Member
Registered: 2022-01-18
Posts: 2

Get colonies every time

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   98oC   60oC   72oC   98oC   58oC   72oC   72oC   12oC
60min 20min  30sec  10sec  2min   5min   10sec  2min   5min    10sec  2min   5min   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~

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#2 2022-01-18 09:00:25

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 130

Re: Get colonies every time

Hi Sekissme,
This is great, thank you for sharing. It looks like three significant deviations from the original protocol that should be highlighted (please correct me if there are more):
1) Lower ratio of insert:plasmid (i.e., 3:1 instead of 20:1)
2) Long 2 min annealing time
3) 'Final touch' cycle
Do you feel like you have sufficient data to create a figure for each of these improvements? I've been out of the lab for many years now, so haven't been able to make any technical advances relating to RF-Cloning. If what you claim is well supported, it could be of real value to the community. And possibly publication worthy? Or has there been a new publication(s) that already details all of these?
Again, thank you so much for sharing. This is really exciting.
-Steve

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#3 2022-01-24 08:16:23

sekissme
Member
Registered: 2022-01-18
Posts: 2

Re: Get colonies every time

Hi Steve,
I had to mention that although a column-purified quality of primer could also work, but a PAGE quality primer would be better on PCR procedures and correct cloning rates in sequencing.
As for the data, it was frustrating at that time, so I run very different conditions almost every day and messed around actually.
The lower ratio of insert:vector (3:1) was advised by my college who had done a RF-cloning with this ratio. Furthermore, this would reduce DNA usage, since my gel-purification DNA recovery was really low. On the other hand, double or triple the amount of vector did give me more vector-insert colonies.
The long 2 min annealing time was my mistake to set annealing times as long as extension times, but the results gave me from all vector colonies to 1 vector-insert colonies out of 5 randomly picked.
The idea of the final-touch cycle was based on a RF-cloning paper that indicate 18 cycles in 2nd PCR would be fine for good results. In addition, E. coli would take methylated DNA as template for proof-reading. Therefore, I added 3 cycles after DpnI digestion to use methylated DNAs (digested vector by DpnI) as new primers to create long methylated DNAs base on vector-insert fused DNAs.

Although I already done a lot of project by this method, sharing procedures is one thing, publication is another. I have to discuss with my BOSS...

Best,

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