RF-Cloning.org Forum

tgattegno

Member

From: Israel

Registered: 2015-03-01

Posts: 2

Secondary PCR does not work

Dear all,
I am trying to clone a 3000 bp insert into a 5000 bp plasmid .
Primary PCR has hardly worked, but I got 400 ng of mega primers for the secondary PCR .
Secondary PCR did not work (2 step ) , I used 5 min extention time. 1:20 primer/insert ratio.
I transformed into One Shot TOP10 Competent Cells, 5 lamda of DpnI treated PCR mix.
Positive control worked for the transformation, DpnI treatment of the parental plasmid worked, as I only got one colony.
I will be happy if you could help me improve my Primary PCR yield and help me with the Secondary PCR.
Thank you,
Tamar

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR does not work

Hi Tamar,
I've pulled up your project from the backend (AFF-1, right?), and the template portion of the reverse primer is very AT rich. I expect that has something to do with your 1° troubles. Maybe try a touchdown protocol to try and increase your yield. You might need to order new primers though, if yield doesn't improve, with longer template binding regions.
Alternatively, maybe consider switching to In-Fusion.
-Steve

tgattegno

Member

From: Israel

Registered: 2015-03-01

Posts: 2

Re: Secondary PCR does not work

Dear Steve,
Thank you for your help.
Is the three step Secondary PCR preferable for my project ?
Or should I stay with the two steps with longer primers?
Thank you,
Tami

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR does not work

If you are able to recover enough template, I would attempt it both ways. Otherwise, I would probably stick with the two step.
I've got my fingers crossed for you.
-Steve

shieyien

Member

Registered: 2015-02-10

Posts: 3

Re: Secondary PCR does not work

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?

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR does not work

Hi Shieylien,
Your protocol looks fine to me, and while the poly-A would make me nervous, I would expect its biggest impact to be on primary PCR yield, not the secondary (although you are probably at higher risk of the final insert being a little different than expected). Definitely try the 3-step method, and if you can get hold of some high competency cells, I'd try those. The fact that you have blue-white screening is huge, because even if there is something weird going on and you get lots of negative clones, at least you'll be able to see the few that maybe worked.
Good luck,
-Steve

shieyien

Member

Registered: 2015-02-10

Posts: 3

Re: Secondary PCR does not work

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

shieyien

Member

Registered: 2015-02-10

Posts: 3

Re: Secondary PCR does not work

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

Last edited by shieyien (2015-09-15 09:31:25)

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Secondary PCR does not work

Thanks for the update Shieyien. There have been some projects in the past that I've needed to screen upwards of 70 colonies as well, so I feel for you. I'm really glad you were able to get your construct made though, congrats.
Now go do awesome things with it
-Steve

ps. I think the overnight DpnI digest is overkill. If you were to do a controlled experiment, I doubt that you would see any difference in the amount of parental plasmid between a 2 hour digestion and a 12 hour.