I'm doing the mutagenesis process, but i dont get it

[mutagenesis]

I'm working on mutagenesis with deletions in the OS9 plasmid.

So I run a pcr to cut its specific area, do a transformation, wait overnight for cells to grow, pick colonies, rotate overnight in agar, miniprep or maxiprep, and then run the dna gel electrophoresis.

but what am I actually doing? I don't really understand what im doing. Can someone explain each step and why I do it?

[jiajia1987]

I suppose you are performing a deletion PCR (you need to confirm this with me)? If this is the case, you can refer to:

PCR-mediated deletion of plasmid DNA
by: M. Hansson, K. Rzeznicka, M. Rosenback, M. Hansson, N. Sirijovski
Analytical Biochemistry, Vol. 375, No. 2. (15 April 2008), pp. 373-375.

After the PCR, transformation is done so that the plasmids (with the deleted portion) can be replicated inside the bacteria. The bacteria is grown on agar plates and colonies (from bacteria that has been successfully transformed and which contains plasmids with the desired antibiotic resistance) are picked to grow in broth. This broth is incubated overnight, following which a miniprep or a maxiprep is carried out to isolate the plasmids with the deleted portion.

I only told you very briefly for each step. If you need more help, just ask more specific questions for us to answer. =)

[Viv01]

Hi,

I Need to introduce to Restriction enzyme sites (Sal1 & BamH1) for my plasmid. But first I am trying to introduce a single mutation by using sal1 RE specific Primer in a single pcr reaction. But I could'nt able to get any colonies. I use stratagene kit. The conditions are given below:


PCR Cycles



95 C for 1 min 1 cycle only

95 C for 50 sec

58 C for 1 min 12 cycles

68 C for 20 min

68 C for 15 min 1 cycle only



Plasmid Size 4.7 kb

Insert Size 2.6kb

ORF 1431 bp



Primer length used - 48bp



Template original con: 50ng\μl

But con used: 2.5ng\ μl ( 20 times dilution)
I'd really appreciate if you can help me.

[JLeo]

I'd try two things off hand: lower your Ta to 55 degrees, and use more template because the mutagenesis PCR is linear, not exponential (I assume you are just using 2.5 ng total in the reaction? You could go up to 25 ng). If this doesn't help, check your primer sequences to make sure they are correct and have a high enough Tm.

[Viv01]

Thank you for ur suggestion.. I will try it and get back to you