problem with pGEMT (View forum version)



deespike

Posted 07 January 2010 - 06:40 AM

hi all

I have been trying to clone my 1.6kb pcr product into pGEMT for quite a long time but finally achieved no true colonies.I always get white colonies but no ligated product is there only pGEMT.My blue white selection works but I donot get any ligated sample?
Can anyone help?

thanks in advance

almost a doctor

Posted 07 January 2010 - 07:19 AM

hi all

I have been trying to clone my 1.6kb pcr product into pGEMT for quite a long time but finally achieved no true colonies.I always get white colonies but no ligated product is there only pGEMT.My blue white selection works but I donot get any ligated sample?
Can anyone help?

thanks in advance



When you say your blue/white selection works what do you mean exactly? As far as I know the empty vector should give you blue colonies, so if everything is empty... howcome you have white ones?

More information on your protocol will help us help you, for example, how do you prepare the insert?

gauden

Posted 07 January 2010 - 09:34 PM

hi all

I have been trying to clone my 1.6kb pcr product into pGEMT for quite a long time but finally achieved no true colonies.I always get white colonies but no ligated product is there only pGEMT.My blue white selection works but I donot get any ligated sample?
Can anyone help?

thanks in advance


Do you know that there are some white colonies which are settle of the blue colonies?. I think in the case you keep the indicator plate for a long time.
If not, you have to calculate the ligation ratio. Good luck to you

gauden

deespike

Posted 08 January 2010 - 09:10 AM

I am using gel purified pcr products in molar ratio of 3:1 insert:ector ratio . how much time should i keep my amp plates?i keep for 14hrs.