Ligation Failure
I am constructing plasmid using restriction enzyme (RE) digested plasmid and RE cut DNA fragment
1. Designing primer for each fragment and PCR; then run gel obtain desired band.
2. Use TOPO Zero blunt cloning kit then use Miniprep kit obtain whole plasmid: each sequence of different fragment is correct (checked by sequencing)
3. Digest plasmid and cut each fragment from plasmid with RE then run gel and extract gel to obtain correct band (extracted DNA product stored at -20 degree)
4. Ligate RE digested-plasmid (2319bp, containing AmpR) and fragment (this fragment is from DNA string fragment, 5’ to 3’ arm, after digestion it is 682bp) using ligation kit at 16 degree, 20 min (kit recommended), vector: insert ratio =1: 1; then transform into DH5alpha.
5. Do Miniprep and analyzed by sequencing: Failed, no insertion in plasmid
I think the digestion is successful because I’ve confirmed with gel electrophoresis then gel extract plasmid and fragment (also confirmed by gel electrophoresis, the bands are all clear); the sequencing results showed no sequence between two RE sites in the plasmid. I’ve tried
1. Extend ligation time, 20 min to I hr
2. Higher vector: insert ratio from 1: 1 to 1: 3. But the concentration of extracted plasmid and fragment is low (Plasmid: 30 ug/ml; insert: 7 ug/ml) even I’ve extracted several pieces of gel in one column. Quality is not good either. I’m not sure if I should do purification since the extracted fragment is not much and low concentration.
3. Now I’m trying new ligation mix.
I couldn’t figure out what caused failure of ligation. Is it possible that digested plasmid ligate itself so it cannot be inserted? Since the sequencing went well and the peak was clean, should I try to purify gel extracted product? I didn’t use fresh digested and extracted fragment, should I use freshly before keeping them at -20?
Hi there,
If the transformants contain empty plasmid only it means they result from either plasmid self ligation or undigested plasmid. In both cases it's an issue with digestion efficiency so you have to make sure double digest is 100% and that double digested plasmid is gel purified to get rid of small bit of DNA which could facilitate empty plasmid recircularization (gel purification is also advised for insert).
I also suggest to systematically run controls for ligation with digested plasmid alone and digested plasmid alone with ligase. The first will tell you about non digested plasmid in the ligation mixture and the second will tell you also about self ligation frequency during ligation. These are needed to know where the problem lies and if the ligation is actually successful.
To confirm, are you using a different restriction enzyme for each end of the fragment? Also I would possibly look at using another gel extraction kit......if you can't obtain a good enough fragment concentration when using multiple gel excisions then the kit itself may be an issue. The TOPO cloning reaction is very efficient and forgiving and may be able to accomodate a level of contamination would be inhibitory in a regular ligation protocol.
You can cut out a LOT of these steps if you used a TA TOPO cloning plasmid and an PCR enzyme that leaves an A overhang. No need to do all of the digesting, purifying, gels, etc. You will lose some of your product every time you do a cleanup step.
Also, your concentrations (assuming you mean micrograms per microliter and not milliliter) are still way too low. Use more of your PCR product for your RE digest (if you don't just use a TA TOPO plasmid). Run out a SAMPLE of your digested PCR product if you really want, but you can honestly just skip that step. Don't try to do the RE digest, then run out ALL of your PCR product, then gel purify it. You will loose way too much of your desired product. Waste of time and reagents.
Short version, just buy a TA TOPO cloning plasmid and directly ligate in your PCR product. Skips all of the other steps and it works really, really well.
Your protocol is very similar to mine and it normally works fine. Even with that low concentrations.
I'm wondering, if you use a phosphatase in your digest? Ligase can only ligate, if one of the DNA strands used, contains a 5' phosphate group. Using a phosphatase in the vector digest removes the phosphates from it and thereby hinders selfligation (if all enzymes perform at 100%). Normally you still have some background of selfligation, but it is way less than without phosphatase. Which obviously increases the change for correct insert ligation or at least picking the correct clone. Just make sure, that you ONLY dephosphorylate the vector and keep the phosphates on your insert.
I use antarctic phosphatase from NEB and you can just add 1 uL of it to your restriction digest of the vector without worrying about buffers/concentration. That might differ for other phosphatases, obviously.
Dear all:
I've fixed this problem. Really really appreciate your suggestion!
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