I am trying to remove the EGFP insert from pEGFP-N1. I cut the insert using incompatible rest. sites and religate using gap filling PCR and standard ligation reaction. But the transformation on Kanamycin plates does not give any colonies.
Hi Nahla,
possibly your gap filling PCR is not working: maybe you have the wrong overhang (you need a 5' overhang to fill the gap with a DNA polymerase) and the Taq polymerases also adds overhanging As at the 3' end. Typically one uses the Klenow fragment or T4 polymerases to fill in 5' overhangs. With 3' overhangs it's more complicated. If you don't have specific sequence and restriction sites specific requirements, why don't you keep it simple and easy and just blunt your endings (whatever they are, 5' or 3' overhangs) using the Mung Bean nuclease?
https://www.neb.com/products/m0250-mung-bean-nuclease#Product%20Information
Also, make sure your ligation protocol is the correct one: for blunt end ligation it is best to incubate O/N at room temperature.
Good luck.
Perhaps you could tell us which restriction enzymes you are using? Just to check if you are cutting out some other essential plasmid bits...
Also, I would assume you want to put something into where the EGFP is why else would you need this vector? I'm confused why you are not digesting your plasmid vector, gel purifying and then ligating in your insert?
Actually, looking at the EGFP-N1 plasmid map
https://www.addgene.org/vector-database/2491/
and
https://www.neb.com/tools-and-resources/selection-charts/compatible-cohesive-ends-and-generation-of-new-restriction-sites
you have NheI (G/CTAGC) and XbaI (T/CTAGA) before and after the EGFP; these enzymes generate compatible ends, so you could cut with those, gel purify and then religate. You could even check success by recutting the newly formed restriction site with BfaI.
Thanks Pietro, I am using phusion high fidelity taq. But I will try the ligation in RT.
Petra thanks alot.
Xba did not work with me.for some reason but I will definitely try it again with NheI.
You have to digest with both XbaI AND NheI as they do not cut the same sequence. However, after the cut they will leave overhangs that will stick to each other.
to find the right buffer to use:
https://nebcloner.neb.com/#!/redigest
but I would also do a control with single XbaI digest and a no enzyme control.
If XbaI does not work then you need to buy new enzyme.
These are all very basic things - is your supervisor not available for questions?
yes I used xbaI with bamhI but it did.not work
When I used bamhI and notI it worked,but the transformation did not give any colonies after blunting and ligation.
when you say "it worked" or "didn't work" I assume you put the digest on a gel?
ah maybe you didn't take into account that XbaI is blocked by dam methylation?
https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation
To get around it you'd need to grow your plasmid in dam- E. coli such as DH5 alpha (which is what I do habitually).
Yes I ran it on a gel. Yes I found out about the methylation but some sites say it is and some say,it isnt.
Anyways I will use the bam and not again and optimize in the ligation. Thanks alot for your help.
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