I am trying to ligate my insert (1700bp) into pET30b vector (5.4kb) with Nde1 and Xho1 restriction site in order to use N terminal histag purification. The 1: 3 ratio of vector (50ng) to insert (250ng) were incubated at 23°c for three HRS using the invitrogen T4 DNA ligase. the digested vector and insert were eluted after double digestion and directly used for ligation without making dilution of them for your kind information. I have added 5µl of the ligation mix in to 50 µl of BL21 DE3 cells. When I screened through blue white selection, it was appeared blue colonies, some time the lack of stop codon in the insert will happen like blue colony? However, I was tried with pET forward and reverse vector, there was no desired amplification at 2kb (1700 my gene + 300 for pet vector).
Hi there,
DNA fragments should be gel purified to remove unwanted digest products or non digested products.
The ratio you have to consider in ligation is molar ratio which basically is comparing the concentration of compatible ends of vector and insert. Ratio1:3 means there are three times more inserts extremities than vector's. It is not the case from the amount you have used: 50ng of 5.4kb vector and 250ng of 1.7kb insert is ratio 1:15!!!
Considering 50ng of your vector in ligation, the equimolar quantity of insert would be 15ng as the insert is roughly 3.2 times smaller than vector (5.4/1.7=3.17). So for a 1:3 ratio, you will need roughly 45ng of insert.
You should run ligation controls (vector alone to measure non digested vector and vector plus ligase without insert to measure self ligation event).
pET30b vector is not a alpha implementation vector and BL21(DE3) is not a lacZΔM15 strain. E. coli transformed with a vector with or without your insertion will all be blue. Simply pick and grow a few single colonies, miniprep the plasmid DNA, check the insertion with NdeI and XhoI, there must be some clones with your insert!
To design the primer carrying the NdeI site, you have to put at least 6 bp to the end to insure the enzyme digestion efficiency, otherwise, this site is not easy to be cut. One alternative way would be ligate this fragment into pBlu2SKP with XhoI and SmaI sites by digesting pBS with these two enzyme and digesting your insertion with XhoI. After you obtained enough the consequent plasmid, you can digest the consequent plasmid with these two enzymes, good luck!
Sorry for my late response . Thanks for all your suggestion sir. I had tried ligation 1:3 (50ng, Insert 47ng) and 1:5 ratio (vector and 50ng vector, 80ng insert). I got colonies in 1:5 ratio ligation. I proceed with colony pcr got non specific amplification and isolated Plasmid DNA and gone for pcr with vector specific primer got amplification around 3kb, expected size 2kb. followed by restriction digestion. there is no insert release. I don't know why i am getting non specific amplification? if there is no insert it should contain self ligated vector. Here i attached the gel picture.
lane 1- 1kb DNA ladder
lane 2&3- pET30b plasmid for control
lane 4-20- colonies from transformed plate (1:5 ligation ratio)
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