RF-Cloning.org Forum

ebeisn2

Member

Registered: 2014-01-23

Posts: 1

any ideas on what I am doing wrong?

Hi, so I was just introduced to this technique a couple of weeks ago, and I am having no luck getting it to work.  I have sequenced my plasmid and my desired inserts and everything is as expected.  I have made the primers according to this website and I am able to amplify the genomic DNA with those primers to get my desired insert product.  I have tried both the 2-step and the 3-step secondary PCR using the extension times recommended by this website.  After DpnI digest (I have done both 3 hrs and O/N), I electroporate them into a DH10B strain, recover in LB and then plate on selective media.  When I do colony PCR the next day using the vector primers (T7 Promoter/T7 Terminator), 20+ colonies, all of them are negative for insert.  Instead, all of the colonies carry the original plasmid sequence.  I have also digested the same amount of plasmid with DpnI without running PCR on it, and I get no colonies, as expected.  All of these attempts have used the same batch of electrocomp. cells.  Is it possible that I am somehow amplifying the plasmid?  Any help would be appreciated!  I would love to get this method up and running in our lab.

Thanks!

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: any ideas on what I am doing wrong?

Hi there,
You have the distinction of being the very first (non-russian spam-bot) person to post in the forum. Thanks for making my day
What is the project ID? I'll have a look and see if anything jumps out at me.
-Steve

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: any ideas on what I am doing wrong?

Thanks for PMing me the project IDs.
I can see right away why you're not having a lot of success. The plasmid portion of the primers you've designed are extremely high GC, and when I plugged one of them into OligoAnalyzer, it gave me a nasty (-33 kcal/mole) dimer. I'm looking at the plasmid you're using, it seems like you're cloning right into the (admittedly sparse) MCS, so is there a technical reason you've chosen not to use the BamHI and XhoI sites?
For your S._suis_ project, I tried shifting the insert sites to 5388-5391, which should retain your reading frame and not break the his-tag or thrombin site. The primers look really good in those positions, and I'm guessing you could do something similar with the remaining constructs. I'm not sure you're going to be able to make your current primers work unfortunately, they are really ugly, and you could spend a lot of time before you have success :S

ebeisn2

Member

Registered: 2014-01-23

Posts: 1

Re: any ideas on what I am doing wrong?

Thank you for your help.  I knew the plasmid portion of the primers might be problematic.  I am trying to clone these genes in the exact same position in the plasmid as a previous post-doc did with a different variant of the gene.  She used NdeI/BamHI sites for insertion, but unfortunately, the Bovis variants have NdeI sites in the middle of the gene.  I will take a look at the alternative site that you recommended and see if I can make it work for our purposes.  I guess I could always try to inverse PCR a different restriction site instead of the NdeI site, but your method looked so appealing.  I hope that I can get it to work at some point!

As far as the GC content in the plasmid-primer portion - at what Tm should I stop considering the primer as compatible?  70C?  These genes are all surrounded by GC-rich areas upstream, and GC-poor areas downstream, so the GC content difference also appears to be a problem for designing primers.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: any ideas on what I am doing wrong?

Tough break about the restriction sites in your insert. I've fought with that in the past too.
I'm almost certain that the insert sites I picked would give you good results, and I would be willing to bet that it's not going to change your protein enough hinder your experiments. I'm assuming you just want to purify the product for IPs or something, so you can probably get away with it. As for the Tm, that isn't really what matters so much, as does the presence of dimerizing sequences. In the future, I would suggest that you check both sides of the primer before committing to synthesizing. I've been toying with the idea of including a new tool on the site that will analyze the primers, but I just haven't sat down to implement it. One day.