For the past few months, my aim was to insert 4kb long gene currantly found in pBlues into pUAST, using 2 restriction enzymes: NotI and XbaI. In the begining my insert seem to logate back to the template, therefore I used all different strategies to purify my gene )eg. gel extraction). With this I obviously loose a lot of DNA, but not completely. Following the ligation and transformation: I , now, do not have any clones. I would appreciate if anyone who did similar work, or already has the experience working with pUAST, give me some feedback.
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george@CASE
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Posted 28 March 2005 - 08:49 AM
I would advise you to gel purify your insert once you cut it out of pBluescript.
If you are having problems with DNA yield, increase the amount of DNA in your digestion.
I usually use 5 uL of DNA from a miniprep, do a 50 uL RE digestion, then run the whole reaction on 2 lanes on a gel. I then cut out bands from both lanes.
I would use only one gel extraction column for this, you will have to make up tubes to balance once you do the centrifugation steps.
Also, make sure that you are cutting pUAST completely with BOTH enzymes. Since these two restriction sites are close to each other, there is no way to tell that on a gel.
Make sure that the REs are in their optimal buffers. Not I and Xba I don't have the same optimal buffers so you have to work around that.
My general advice to you would be: do not try to cut corners when doing these experiments. You have to do gel extraction, you may even have to do sequential digests.
If you are having problems with DNA yield, increase the amount of DNA in your digestion.
I usually use 5 uL of DNA from a miniprep, do a 50 uL RE digestion, then run the whole reaction on 2 lanes on a gel. I then cut out bands from both lanes.
I would use only one gel extraction column for this, you will have to make up tubes to balance once you do the centrifugation steps.
Also, make sure that you are cutting pUAST completely with BOTH enzymes. Since these two restriction sites are close to each other, there is no way to tell that on a gel.
Make sure that the REs are in their optimal buffers. Not I and Xba I don't have the same optimal buffers so you have to work around that.
My general advice to you would be: do not try to cut corners when doing these experiments. You have to do gel extraction, you may even have to do sequential digests.
