Need help for restriction enzymes - (Oct/27/2009 )

Hi
I got the problems about digest my insert and vector by restriction enzymes. I use BamHI and SalI to digest my vector (pET duet1) and insert genes (double digestion). Then Incubate at 37C for 2 hrs. Is that enough for 2 hrs?. After that I did ligation. Transformation and I got some colonies. Next step I did PCR colony screening but I did not find my expected band on the gel. I think it was because of restriction enzymes did not work properly. So any idea that I can confirm my restriction enzymes work? Please help.

Did you gel-purify your digestions before ligation?

Can you recover plasmid DNA from some of your transformants and digest it, looking for an insert?

Perhaps your PCR colony screening failed -- did you have a postive control?

HomeBrew on Oct 27 2009, 12:32 PM said:

Thank you for your advice
Yes I did purify my digestion before ligation.
I got colonies and I checked PCR colony screening and I got band but it was not the right size about 900 bps. I found band which lower than my expected size. It was false positive one I supposed to say. I don't know what should I do next. Any idea for me please

If your insert does not have an EcoRI or BspMI site, you can do the ligation (but I'd suggest you do 2 hr @ RT, not 37) then digest with the enzyme; this will linearise any single-cut plasmids, but not affect your double-cut, insert ligated plasmids.

To check the REs, just do single digestions and run on a gel. To test the ligase is working and the insert is correctly cut , treat some DNA ladder and some insert and run on agarose - the ladder should shift up and the insert should have at least a trimer-sized band.

When doing colony PCR, pick the colony into some TE and mix well, then add 1 ul to the PCR reaction. This increased the number of successful reactions to >95%.

swanny on Oct 27 2009, 10:28 PM said:

I completely agree. I used to just pick some small amount of a colony into 30 ul of PCR master mix, but then switched to picking about the same amount of colony into 50 ul of sterile water and using 1 ul of this suspension as template. My success rate went up dramatically.

HomeBrew on Oct 27 2009, 07:34 PM said:

Thank you guys I will try and let you know later
Cheers