1 reply to this topic

[jaya2020]

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.
95°C 50 sec
95°C 50 seconds
55°C 1 min

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

I even tried adding DMSO once. Nothing worked.

Any help would be greatly appreciated.

Regards,

jaya

[Kaioshin]

jaya2020, on 16 April 2011 - 03:36 AM, said:

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.
95°C 50 sec
95°C 50 seconds
55°C 1 min

68°C 5 minute x 18 times
68°C 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