9 replies to this topic

[jiajia1987]

Hi people,

Just would like to check if anyone here has done deletion PCRs before.

I am interested in making an inactive Mdm2 (the protein that binds to p53) and I am thinking of using deletion PCR to make an inactive PCR. As this is my first time trying out this technique, I need some guidelines on how to go about doing it. It would be good to discuss this topic here with you all. The Mdm2 is in a plasmid and I am not too sure if it is alright to just use Taq polymerase (I have been using Taq from Bioline all the while for all my PCRs) or is there a need for me to use a high fidelity polymerase such as Pfu?

Edited by jiajia1987, 08 June 2009 - 12:55 AM.

[jiajia1987]

And,
I am unable to get a copy of the following paper. The following paper is what I am looking into as an option for me to produce deletions in my desired gene. Can anyone help to send me the article? It will be greatly appreciated.

PCR Mutagenesis by Overlap Extension and Gene SOE
Vallejo et al. Cold Spring Harbor Protocols.2008; 2008: pdb.prot4861

[Rsm]

Is there already a paper describing inactive Mdm2? If you need a point mutation, I would suggest you use site-directed mutagenesis. Just order a forward primer (30mer), consisting of 14nt before your mutation, then mutation and another 15nt after mutation. Reverse primer is complementary to this.
Deletion PCR works similarly, 15nt before deletion and 15nt after, reverse is complementary.
I suggest you to use a good Pfu polymerase, not too many cycles (maybe 18), and to add Dpn1 after PCR.
Major problem is design of primer, I haven't found any good program yet... you might need to check different annealing temp and/or primer...
Hope this helps,

Minna

[jiajia1987]

Minna, on Jun 9 2009, 10:16 AM, said:

Is there already a paper describing inactive Mdm2? If you need a point mutation, I would suggest you use site-directed mutagenesis. Just order a forward primer (30mer), consisting of 14nt before your mutation, then mutation and another 15nt after mutation. Reverse primer is complementary to this.
Deletion PCR works similarly, 15nt before deletion and 15nt after, reverse is complementary.
I suggest you to use a good Pfu polymerase, not too many cycles (maybe 18), and to add Dpn1 after PCR.
Major problem is design of primer, I haven't found any good program yet... you might need to check different annealing temp and/or primer...
Hope this helps,

Minna

Dear Minna,

I didnt know that there is a paper describing inactive Mdm2. I am new to this field, do you mind telling me which paper is it? I can then try to find it in the databases.

And, I am using deletion PCR to remove a stretch of region because I need to remove the whole region that binds to p53 due to the nature of my work, so I can't use site-directed mutagenesis.

I am thinking of using Pfu too, hopefully my supervisor will provide me with it. My template is in a plasmid and I've heard that about 20 cycles is the max. Why cant we use a longer cycle?

[jiajia1987]

I just had a talk to my supervisor and the proposed plan that I had in mind is kind of different from his. He is thinking of a deletion PCR where the primers are non-complementary and are on either sides flanking the deletion site. The PCR product will be a blunt-end product and I can use phosphatase and kinase to ligate the ends together. The thing I don't understand is. WHy would we need a phosphatase or a kinase to ligate the ends together? He mentioned something about primers coming with (or was it without, he spoke too fast) phosphate groups at the 5' end. Does anyone have any idea which is which? The ligated product will be a plasmid with the deletion and this can be transformed into E.coli for sequencing purposes. Now, I am confused as to where the Dpn1 digestion comes in.

Help please?

Thanks in advance.

[Rsm]

Hi there,
I don't know any point mutations of Mdm2 resulting in a dominant-negative effect. It seems most people are using deletion mutants. Anyway, trust your supervisor on this, he has more knowledge about Mdm2 that I will ever have.
I think your plan might work as well. You'd need to have your primer phosphorylated at the 5' end, make a PCR with Pfu, purify and ligate with T4 ligase (not phosphatase or kinase). You'd need to add Dpn1 during ligation to remove the old unmodified plasmid (Dpn1 cuts only methylated ie. old plasmids).
20cycles are recommended because Pfu has exonuclease activity, the more cycles you make the shorter primer you'll have. That might result in unspecific amplification.
Good luck!

Cheers,
Minna

[jiajia1987]

Dear Minna,

Thank you for explaining to me the molecular biology side of things! It definitely makes a noob like me understand things so much better. If only there is a live chat here so all of us can just chat online and clarify our doubts!

[jiajia1987]

And by the way,

I have read that Pfu enzyme extends about 2kb/min. The region I am amplifying on my plasmid is about 7kb. so that gives 14mins. But my supervisor was saying 8mins shud be enough and he mentioned Pfu Turbo. Is this a normal Pfu enzyme or some faster enzyme coz of the name 'turbo'?

[Rsm]

If the Pfu amplifies 2kb/min and your plasmid is 7kb, then you'd need 3.5mins for whole amplification, not 14mins... Anyway, usually you use 1min extension per 1kb of plasmid. I have used Pfu Ultra with very good results, don't know about Turbo.
Cheers,
minna

[jiajia1987]

ooooooooh!!!!!!! that was a miscalculation on my part. thanks for pointing it out!!!!!!!!