RF-Cloning.org Forum

ZacJ

Member

Registered: 2014-08-12

Posts: 4

2nd PCR not working

Hi there,

I really like the RF cloning protocol and have been trying to swap the V5/His tag in the pEF1 vector with an mNeptune sequence. My first PCR works really well and I get good yield from the gel extraction but when I try the 2nd PCR I get no colonies at the end. The 2nd PCR product is treated with DpnI which must work as I don't get any false positives. I have tried both the 2-step and the 3-step with 60 degrees annealing temperature but to no avail. The only thing that I can think of is something to do with the plasmid annealing part of the megaprimer. I'm not sure whether the details provided by the website are on record somewhere...

I hope you can help.

BW,
Zac

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: 2nd PCR not working

Hey Zac,
I think I was able to find your project from the backend, and on first glance everything looks good. The plasmid is a bit big, but by no means horrible. What were the details of your transformation?
-Steve

ps. I've linked the project I think was yours to your account. Go into the project management dashboard and see if it's right.

ZacJ

Member

Registered: 2014-08-12

Posts: 4

Re: 2nd PCR not working

Thanks for the quick reply. I tried to find the project on plasmid management after going through the rf-cloning.org home page but couldn't see it. The construct name was pEF1-mNeptune.

I transformed the DpnI digested PCR into OneShot MAX efficiency DH5a chemically competent cells from Life Tech. using the standard 42 degrees heat shock protocol. Shook them for half an hour in SOC at 37 then plated the whole volume onto two LB+Amp plates

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: 2nd PCR not working

Try looking in your project management dash again; I made a mistake, but it should be there now.

I'm guessing you had a positive control that came up as expected? And did you use the entire PCR reaction in the transformation, or just 1 μl (i.e., 1 μl good; 20 μl bad)? I'm surprised you didn't get any negative colonies at all using the OneShot Max cells, because they are crazy sensitive.

ZacJ

Member

Registered: 2014-08-12

Posts: 4

Re: 2nd PCR not working

Yep had another look in the project management section and it's there thanks

I didn't have a positive control in the last transformations I tried for RF but I used the cells from the same batch for a regular Maxi prep and they worked fine and the transformation protocol hasn't changed. I should probably include a control in subsequent transformations just to rule it out.

I used the entire PCR reaction to transform. Was that correct? I assumed that as it is a rare event it would be better to use all of it...

There was absolutely nothing on either plates and I know I've got the right antibiotic.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: 2nd PCR not working

Hey again Zac,
Before you do anything else, redo the 2° PCR and only use 1 μl of the reaction in the transformation. You've made a common mistake by assuming 'more is better', but you're changing the salt concentration of the comp cells and significantly reducing their efficiency. You should not exceed 5% of the total cell volume when adding DNA, 10% max. The cells are actually sensitive to total DNA as well (20-50 ng is a common target for high efficiency), and there is probably 10-100X too much total DNA (albeit chewed up by DpnI) in the full PCR reaction.
One other thing I've noticed is that you appear to be cloning into the MCS, and there are some really ugly dimers on the plasmid side of both primers. Bump up your annealing temp to 68°C, and you might want to increase your extension time to 3:30 min.
Good luck, and let us know how it turns out
-Steve

ZacJ

Member

Registered: 2014-08-12

Posts: 4

Re: 2nd PCR not working

Hi Steve thank you for the reply. I repeated the 2nd PCR with both the original protocol and with the increased annealing/extension time but only using 1 & 2 ul of the PCR mix after digestion and it all worked perfectly The higher annealing temp gave me more colonies so you were right about the dimers. I really appreciate you guiding me through this and definitely know better for any future RF experiments.

Thanks again!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: 2nd PCR not working

Congrats! I'm glad it all worked out.
Take care,
-Steve