2 replies to this topic

[caren]

I had some problem about Site directed mutagenesis(SDM). Anybody can help me,please.

I used quik-change Lighting Site directed mutagenesis(SDM) kit to did mutant(single amino acid change).

This is my primers:

N164A _F   5'-ggacttatcactccctgggcttatcctctgctgatggc-3'
N164A _R   5'-gccatcagcagaggataagcccagggagtgataagtcc-3'

I used program of STRATAGENE to design primers:
http://www.stratagen.../qcprimerdesign

Base length of each primers were 38 base  and  Tm of my primers were 78.97°C. The primers had GC terminate. The mutation is balanced as they are exactly in the middle. Everything is OK.

My  plasmid+insert size is about 6.8 kb, I strictly follow the protocol. when I ran agarose gel after cut with DpnI, I saw many PCR product and its expect size correct.


But I don’t have any colony form  transfromation of mutant into XL10-Gold  ultracompetent cell. So I used my own completent DH5-alpha to did Heat-shock and I didn’t have any colony.  I did Electrophoration using DH5-alpha for transfromation instread and this method I didn’t get colony again -*-

All method I added PCR product 2ul.

For control in this kit-pWhitescript- I did it already and it had strong band PCR and got many colony when transform into XL10-Gold  ultracompetent cell.

I need to know which anybody has other efficiency transformation method to transform my product.


Anybody can help me,please.


Caren

[Stuck with Ligation]

I have had mixed luck with the site directed kits. The TM's seem a bit high. I always aimed for Tm's ~63-65. It sounds like your cells are competent which is good. You can also try using 3-5 uL of the PCR mixture. Also, you may only see 1-4 colonies after transformation which is normal with the quickchange kit. I have never run my quickchanges on an agarose gel so I cannot help you there.

Good luck.

[caren]

Stuck with Ligation, on Nov 20 2009, 03:05 AM, said:

I have had mixed luck with the site directed kits. The TM's seem a bit high. I always aimed for Tm's ~63-65. It sounds like your cells are competent which is good. You can also try using 3-5 uL of the PCR mixture. Also, you may only see 1-4 colonies after transformation which is normal with the quickchange kit. I have never run my quickchanges on an agarose gel so I cannot help you there.

Good luck.

Tm of primers for this kit must 78C up, aren't it.
I'll add PCR product 1ul, 2ul, 3ul, 4ul into completent cell.
I used LB-broth for transformation.
May be the problem from my broth,What do you think?

I'll try it agian.
Thank a lot