I'm using pACYC vector amd I'm doing double digestion with BamHI and HindIII for both of my vector and insert.
1) In my primer design, I only added one base pair before the restriction site sequence. Will this affect my insert digestion efficiency?
2) I did single digestion for my vector to test if the restriction enzymes are working properly. When I saw that each enzyme works well with my vector, I did double digestion using FD-BamHI and FD-HindIII using the universal buffer of thermoscientific. This double digested vector was then ran on gel, purified and the concentrations were in between 7-35ng/microliter. What could be the problem? I double digested it at 37degC for 45-1hour.. I cannot exceed one hour because BamHI will exhibit star activity. as indicated in its specification..
3) I did ligation with a 1:5 molar ratio vector:insert. I also ran my ligation mixture on the gel and I got 5 bands (sizes): a very thick band for my insert alone, a very thick band of my dimerized insert, a very faint band of my plasmid alone, a very faint band of my plasmid+insert, a very faint band of supercoiled plasmid. I transformed them and I already picked 45 colonies and all were NEGATIVE.
4) I did Colony PCR.. On my first run, everything was ok, negative control was negative on gel, positive control was positive on gel yet all my picked colonies did not show any band. On my next set of picked colonies, all my colony PCR tube, even the tube without template added showed positive band on the gel and its band was the size of my insert. So I repeated the 2nd set of picked colonies and I got positive band for my positive control, no band for my no template added tube, a very faint band of my plasmid from the negative tube (with plasmid only), and all negative for the picke colonies..
3rd set of colony PCR - negative control showed a faint band of plasmid, No template tube showed no band, positive control tube showed positive size of the insert, all of the picked colonies showed positive result.. So of all the positive tubes in the colony PCR, I chose to grow only 2 colonies for confirmation using digestion. However, my digestion showed negative results. So what was wrong after all?
Why is it that I have a positive result in my colony PCR yet it showed negative result in restriction enzyme digestion?
I can't figure out what is wrong with my cloning.
1. Please clone your insert in a normal TA cloing vector or pJET1.2 if using Phusion polymerase.
2. I believe enzymes may not be properly working so for complete digestion digest your vector about 2 ug for about 3-4 hrs and then gel elute. The fermentas Fast-Digest works great without any star-activity. Do the same with your cloned insert and elute from gel.
3. Ligate in a 1:3 molar ration (vector about 40-50ng & Insert accordingly) and transform.
Good luck
first put your PCR product into cloning vector like pGEM-T, second, digest your new plasmid to release the insert e verify the efficiency of the enzymes. Third you can digest you plasmid using BamHI for more than 1 hr, but only if you calculate in you reaction lower than 5% glycerol, the restriction enzymes are on 50% glycerol. Good luck!!
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