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#1 2017-02-24 10:22:09

asilbering
Member
Registered: 2017-02-22
Posts: 4

Secondary PCR not working

Hi Steve,

First of all I would like to thank you for making this tool available and for helping people troubleshoot their cloning projects!
I am running two projects in parallel to replace one gene in a destination plasmid with two different constructs. They are quite similar, so I have treated both equally.
I have used your tool to design my primers and obtain the cycling conditions.
The project ids are:
proj_id=b39e353abb98a39fffb6439cad384ee4
proj_id=ab3791899fa1eb4ae9559e1e0dafc5ee

I run the primary PCR and obtained nice megaprimer bands for both, with a yield of around 200ng/ul for each (DNA quality was not super-high, the 260/230 ratio was 1.47 and 1.23 respectively).
I used phusion and I added 5%DMSO to the reaction. The cycle I used was:
98°C 30"
98°C 8"
56°C 20"
72°C 1'40"
72°C 5'
With a total of 35 cycles

I then set up and ran the secondary PCR with the quantities of megaprimer and cycling times indicated using the two-step protocol. I used Phusion and added 5% DMSO.
The cycle was as follows:
98°C 30"
98°C 8"
72°C 3'50"
72°C 5'   
total of 15 cycles (I also repeated it with 20 cycles to run product on gel, and while I could see the primer band very well, there was only a very faint band with the expected size for the product)

I transformed one shot ccdb survival competent cells and obtained no colonies (my plasmid contains a ccdb cassette, so I have no alternative). I did a positive and negative control for the transformation (destination plasmid alone which gave a lawn, and plasmid digested with DpnI, which surprisingly gave a few colonies). I know these controls are not exactly the same as running the PCR with no primer and not digesting the product, but I hope they will do).

Do you have any suggestions? Do you think that given the characteristics of my project I should try the 3-step protocol? Could it be a problem that the chunk I am trying to remove from the destination plasmid has more than 1kb? Do you think the DMSO could pose a problem?

I would really appreciate any suggestions.

Thanks in advance.

Best regards,
Ana

Last edited by asilbering (2017-02-24 10:25:41)

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#2 2017-02-24 15:50:25

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 102

Re: Secondary PCR not working

Hi Ana,
I'm not too surprised you are running into trouble; these are tricky projects with big insertions, big deletions, and big destination plasmids... You're also fighting a fairly gross -23 kcal/mole worth of complementation in your forward primer. It's not a deal breaker because if doesn't prime, but still, yuck. Otherwise, your PCR reactions all look fine to me, so you're just seeing the effects of a very low efficiency project. If you wanted to keep burning comp-cells, you could cross your fingers and hope to get lucky, but it might be more economical to try the InFusion method. Your nice megaprimer purifications should work great for this, and while I haven't specifically looked, I'm pretty sure you'll have a useful restriction site somewhere in your deleted sequence. If this simply isn't an option, try the three step, but mostly you're probably looking at doing a bunch of transformations (plating everything), and hoping for the best.
I wish you luck,
-Steve

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#3 2017-02-24 16:19:07

asilbering
Member
Registered: 2017-02-22
Posts: 4

Re: Secondary PCR not working

Hi Steve,

thanks a lot for the quick reply, even if it is not too encouraging. I will try both (Infusion and 3-step).
Let's keep the fingers crossed!
Ana

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#4 2017-02-28 13:36:42

asilbering
Member
Registered: 2017-02-22
Posts: 4

Re: Secondary PCR not working

Hi Steve,

I have been looking at the InFusion protocol you suggested and there are a couple of enzymes I could use to linearize the plasmid (I think I could even remove a big part of the unwanted gene if I use two), but the target sequences of my megaprimer would still not be at the ends. Does the InFusion work also under these conditions? According to the manufacturer, the primers have to anneal with the first 15bp in each end, so I was not sure if it could also be used to "replace" a stretch of sequence.

Thanks a lot in advance.

Best,

Ana

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#5 2017-02-28 14:04:34

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 102

Re: Secondary PCR not working

Hi Ana,
Yep! As long as there is overlap between the megaprimer and plasmid on both ends, it should work just fine. Also, cut down the volumes you use in the reaction so you finish with 1-2 μl. There's no need to mix up a 10 μl reaction, because you're only going to use 1 μl in your transformation (saves a lot of money this way).
-Steve

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#6 2017-02-28 14:45:56

asilbering
Member
Registered: 2017-02-22
Posts: 4

Re: Secondary PCR not working

Great! Thanks!

Ana

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