No band is visible with BamHI and XbaI Digestion - (Aug/12/2008 )

Hello.

I am dealing with pBAC4x-1 (5917bp, a transfer plasmid for insect cell system). In its MCS, XbaI and BamHI are located at position 1498 and 1937, respectively. When i tried to digest at this two sites to screen for the plasmid, there is no band visible in my agarose gel electrophoresis. Expected band size is 435bp. The RE i used is from NEB and the condition is exactly same as the suggested condition in NEB website. I repeat it quite a lot time, so pipetting error should not be in consideration.

To check for the presence of those two sites (BamHI and XbaI) in the plasmid, i performed double digestion with EcoRI and one of the RE, which means EcoRI+BamHI and EcoRI+XbaI digestion. Bright band with correct expected size is observed for both digestion. This directly indicated that BamHI and XbaI sites are present on the plasmid.

So, my question is...why BamHI+XbaI double digestion fail? Your experience is highly appreciated.


* Similar condition happened with insertion in between BamHI and XhoI of the plasmid. Screen using other RE sites showed larger band size in comparing with original plasmid, with indirectly meant there is insertion in that area. However, screening and confirming the insertion with BamHI+XhoI itself revealed no band. Why?


Thanks for reading my post

From NEB website, it says using buffer 3+BSA, but XbaI activity is 75% under this condition. Did you add more enzyme or incubate longer? XbaI is sensititve to overlapping dam methylation. Actually, I don't understand this very much. But I also have some problem with XbaI. So, it might be one of the reasons.

I used XhoI quite well. But BamHI is very sensitive to its star activity. So, the concentration of glycerol, the reaction time, pH or other things might also give you problems. If star activity is working, when you extend the exposure time, you would find a lot of small bands.

How about your control? Because sometimes I used BamHI with another enzyme together. I had a control plasmid which had only BamHI site. Once I didn't find the expected band in my sample. and then, I also found that control plasmid wasn't digested. That means BamHI didn't work.

Also have you tried digesting with one enzyme, checking a small volume on a gel for linearization, precipitating the DNA ad then digesting with the second enzyme?

If you do this with BamHI first then XbaI, and the other way around (in parallel) you may be able to identify the problem.

Do you have any sequencing primers that cover the region of interest to confirm that the sites are ok in the parental plasmid?

Thanks for the replies, Uterus and Chris.

I never run digestion with control because i thought double digestion is not a critical process. Maybe i will perform a control next time.

Chris was suggesting me to do sequential digestion to check for the problem right? Thx. This could be a nice method to troubleshoot this problem. Hopefully it works.

I never perform DNA sequencing of this plasmid. However to confirm that the sites are ok in the parental plasmid, i did run double digestion using a third RE (which means EcoRI+BamHI and EcoRI+XbaI digestion). Correct digested band meant both sites are ok right?