5 replies to this topic

[lab_member]

I am using a plasmid where the restrictions sites on the MCS are BamHI and EcoRI. I have read that BamHI don't work well with double digest with other enzymes therefore I did a sequential digestion: BamHI digest --> PCR purification --> EcoRI digest --> PCR purification.

The fragment that is cut out is only 17 bp and it should be in the flowthrough during PCR purification using the QIAquick system.


I did overnight ligation inlcuding a negative control where MQ water was used in place of the insert, 16C using T4 DNA ligase from invitrogen. Then i directly use 1 ul for transformation into 30 ul of electrocompetent Top10 cells by electroporation.

The problem is that I got a lot of colonies in my negative control plate, although less than my other plates.

I know a re-ligation probably occured but does anyone know of anyway to verify the problem. I can only verify digestion for BamHI but not EcoRI since the latter digestion is on a linear plasmid and the cut out fragment is too small. Now I don't know whether to do colony PCR on the colonies since there is so much colonies in the negative control.

Thanks

[phage434]

Make sure you are cutting with both enzymes independently.  One way to dramatically reduce background is  to use pcr to make the vector.  PCR using primers to the vector, containing the restriction sites and a good (6bp) overhang.  PCR, gel purify, cut with both enzymes, purify and go.

[T C]

Hey

Just do a sequential digestion and CIP the vector, even if you have single cutters, they won't religate.

Best
TC

[lab_member]

Thanks a lot. Although I have no time to do these because my deadline is coming near, I will definitely try these methods the next time. Anyway, is a shorter insert (~60 bp) more difficult to work with then a longer insert (~1 kb)....just curious...

[T C]

Nope

Its the other way round....shorter ones are easier to clone.  The clones come in one shot and all the colonies that you screen are clones.  (Well...if u follow the protocol properly

Best,
TC

lab_member, on Mar 25 2009, 01:21 PM, said:

Thanks a lot. Although I have no time to do these because my deadline is coming near, I will definitely try these methods the next time. Anyway, is a shorter insert (~60 bp) more difficult to work with then a longer insert (~1 kb)....just curious...

[MaggieRoara]

I have used BamHI and EcoRI before together. didnt have any problems