7 replies to this topic

[predoc]

I have a PCR product in which I introduce an HA-FLAG tag N-terminally to my gene. I ligate and transform this product. I got a few colonies on LB-amp. When I purify the plasmid, and run it un-cut on the gel, I see a band about the right size. Next, when I digest with the restriction enzymes that I need to use, I see the control plasmid, but only a smear for the plasmids I am testing. What might be going on?

Thanks

[bob1]

sounds like a homework question...

multiple variants?

[predoc]

I don't understand what you mean by multiple variants. If the digests don't work, I should still see the uncut band and not a smear...won't I???

bob1, on Jan 25 2010, 05:12 PM, said:

sounds like a homework question...

multiple variants?

[bob1]

multiple variants of the same plasmid as a result of the PCR not giving full length and/or mutated fragments, thereby giving you several different digests appearing as a smear.

[predoc]

As far as this is concerned, I got a PCR fragment about the right size, I gel extracted it, ligated, transformed. The picked the colonies. Then I check them with a digestion. I get a smear at this point. The control digestion is ok. It is the digestion of the different colonies I picked that is a smear. What could this mean? I I put them on a gel undigested, I see a band.

[predoc]

I re-read what you wrote but considering that the PCR product is gel extracted, what should I do next?

[bob1]

I'd probably just sequence a few colonies and see what comes up.  It should be that you have some with the correct sequence in there.

I take it you are using a proof-reading polymerase for your PCR?  If so, there isn't much else I can think of to suggest.

Edited by bob1, 27 January 2010 - 03:10 PM.

[predoc]

Thanks Bob, I appreciate the suggestions. I am using proffreading pols. 2: phusion and KOD in separate reactions. I have set up another PCR and also sent out 1 clone for sequencing.