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Sorry for my late update... I finally get my clones using Q5 weeks ago. With 2-step protocol.
I did overnight digestion with DpnI, and then transformed into CaCl2 competent XL1-Blue (lab prepared). [4.5ul final product per 100ul cells]. And I did 10 transformations. This time, I skip the Blue-white selection to minimized potential gene toxicity. I did pooled-colonies PCR on ~70 COLONIES and finally I got 2 positive clones tested by PCR.
I sent them for sequencing, only 1 of of the clone contained single mutation. The other clone was just fine.
My conclusion of this protocol:
1) Q5 works for RF-cloning
2) prolong DpnI digestion / increase DpnI concentration may be necessary to reduce the interference of parental clones.
3) High transformation efficiency competent cells really crucial for high success rate. Else, if using general lab prepare cells, increase number of transformation is necessary
4) It's necessary to screen many clones, as the parental plasmids may predominant your progeny plasmids (with insert).
Despite of all these problem, I still will choose RF-cloning method in future.
Thanks Steve
Is is difficult to acquire high competent cells in short time. Our lab is new in molecular cloning, thus specific reagents/consumables are limited.
I will make new stock of my homemade competent cells. And try to run 3-step protocol with multiple Tm.
And of course, I will double check those blue colonies with PCR if they carry insert.
P/S: I did PCR checking on my first attempt of secondary PCR product (after DpnI digest), and the desired band was almost unnoticeable. I was probably due to poor plasmid quality I use that time.
I will definitely try PCR the secondary PCR (of the 2nd trail) to see if there is any insertion too.
Will update soon once I have the updates. Thanks
Dear Steve,
I had designed several RF cloning projects using your software, I am working on one of it (named: pUC19-fragment_6-polyA). I have successfully get the primary PCR out with enough fragments. But failed to get any colony with the appropriate insert.
I using Q5 polymerase instead of fusion. Below are the running condition of my 2nd PCR:
Reagent (Q5 polymerase kit)
5x Q5 reaction buffer ------------4ul
5x Q5 enhancer buffer -----------4ul
dNTP (10mM each)--------------0.4ul
Q5 polymerase------------------0.2ul
H2O-------------------------------3.3ul
plasmid---------------------------2ul (~32ng)
mega primer --------------------6.1ul (~238ng)
Running condition:
98C-----------------1 min
98C-----------------10s ]
72C-----------------90s ]18 cycles
72C-----------------5 min
add 1ul of DpNI, 37C incubation for 2 hours, then followed by 20 minutes inactivation at 80C.
Transform 2ul into homemade competent cells, TOP10F'
The insert will interrupt the lacZ gene, so I made blue white selection of the transformants. Well, I got a few colonies, but none of them contain inserted.
This is my second time doing the 2nd PCR.
Any idea of why I failed?
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