Hello,
I have been trying to ligate a 1.4kb insert in the pENTR D-TOPO plasmid using different molar ratios like 1:1, 2:1, and 3:1 (recommended by NEB) , but every time no colonies are seen. Only once, with a 1:1 ratio, I got 2 self-ligating colonies. I have repeated this experiment 7 times with different conditions such as increasing the ligation time, using up to 30ng of the insert, using some other competent cells, using new kits, but nothing has worked so far. Any advice on what can I be doing wrong? Considering pENTR is a relatively easy cloning vector, I don't know why it fails every time.
Is your 1.4kb fragment generated by PCR or coming from a digested vector ?
Hi Muhammad Abdullah Jauhar
Usually I do my cloning using 100ng of vector and 200-300ng of insert (1:3 or 1:5). You must be sure the full digestion happened because when restriction enzyme recognition sites are methylated, DNA cleavage may be blocked depending on the restriction enzyme and the type of methylation, so check your enzymes and if it happened, it will be necessary to use a Dam- and/or Dcm-negative E. coli strain, for instance GM48 and JM110. Then you should gel purify vector and insert after digestion to remove bits of DNA that are in your samples which might interfere with the ligation. And finally, after adding T4 Ligase, place your microtube 30 minutes at R.T temperature then at 4 ºC overnight. and use fresh competent cell.
Hope it will help.
Jean-Pierre Dangy
Thank you for reaching out. It is coming from PCR amplification and my forward primer contains the magic CACC sequence.
Farshid Moosavi
Thank you for your answer, the insert is in fact coming from the PCR amplification and the vector I am using has blunt edges. It is the standard directional cloning kit provided by Thermo Fischer. The kit says 20ng of the vector is used per reaction mixture.
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