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Weird problem in in vitro mutagenesis


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1 reply to this topic

#1 jaya2020

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Posted 16 April 2011 - 03:36 AM

Hi,

Iam doing invitro mutagenesis for a 5.2Kb plasmid using stratagene kit. I did not get any results with my plasmid, so checked the control in the kit. Unfortunately the control is not working. I have tried many times, but got no colonies. The conditions used were according to the instructions given by the manufacturer.
95C 50 sec
95C 50 seconds
55C 1 min

68C 5 minute x 18 times
68C 5 minutes.

I even tried adding DMSO once. Nothing worked.

Any help would be greatly appreciated.

Regards,

jaya

#2 Kaioshin

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Posted 17 April 2011 - 02:18 PM

Hi,

Iam doing invitro mutagenesis for a 5.2Kb plasmid using stratagene kit. I did not get any results with my plasmid, so checked the control in the kit. Unfortunately the control is not working. I have tried many times, but got no colonies. The conditions used were according to the instructions given by the manufacturer.
95C 50 sec
95C 50 seconds
55C 1 min

68C 5 minute x 18 times
68C 5 minutes.

I even tried adding DMSO once. Nothing worked.

Any help would be greatly appreciated.

Regards,

jaya


Call the company that you bought it from or invitrogen and complain. If the positive control isn't working and you're following their instructions, then it could be a bad lot/item. The only thing that I would suggest that is under your control is checking your competent cells that you were transforming into to make sure they aren't the root of the problem. Unless, of course, those came with the kit.

You'll likely just get a new kit from them and have to return what you have left of the one you bought




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