Hi everyone!
I have a problem with e.coli cultivation.
I use pET28a and pBHA plasmids. My gene is in pBHA plasmid
pET28a consists of NdeI and XhoI restriction sites.
An insert also has the same restriction sites.
After restriction electrophoresis shows restriction products:
Gene approx - 400-450 bp ( the gene mass - 420 bp)
Restriction product from pET28a - 80 bp (as it is described in standard manual)
After ligation of a gene and an uncircular pET28a the electrophoresis shows products of ligation that look like products of polymerisation of (1) insert (Gene =420 bp, but the major ligation product - 2000 bp by using MassRules DNA Ladder Mix as a marker) and (2) the vector (near start position).
No product in an expected area of 5000-6000 bp persists.
No colonies after 16-20 hours of incubation. Only the positive control (non-restricted circular pET28a) gives many colonies.
I've found that the gene consists of 5-CATATG-3 fragment - classic NdeI restriction site, and 5-GTATAC-3 - the opposite fragment.
The problem is that:
The process of restriction persists (you may see a gene next to 400-450 bp, and previous pBHA vector aprox 2000 bp)
When I use a non-restricted circular(!!!) pET28a it grows! When I put it in a ligase buffer and do not inactivate ligase it also grows!
Negative control (restricted pET28a) is negative.
Thats why I have some questions:
Where can be the problem?
Is it possible, that NdeI that cuts fragments 5-CA'TATG-3 may do it in case of 5-GTAT'AC-3?
It may explain why vector do not react with insert in case of another unusual NdeI site
Hi,
I think this is a ligation problem, try to ligate the restriction fragments from pBHA, you should get something. Or try another restriction enzymes as a control.
Hi there,
Definitely a ligation problem:
do you check ligase activity (in a self ligation assay with single digested plasmid)?
do you gel purify the linear vector?
NdeI is definitely unable to cut anything else than 5'CATATG3'...
Dear Airat R. Kayumov, Nagendra Nath Das, Dominique Liger
Thank you for your help!
The Ligase was changed with the fresh one.
About pBHA - I did the reverse action (gene into the pBHA) and the result was negative - no colonies.
I purify the vector with Gel extraction kit.
As I see there are changes in a gel. The process happens, but not in a proper way. That means that the ligase works but the cause of the problem still remains unknown.
I've changed an insert and a vector ratio and concentration; ligase concentration; temperature (4-14-25
1-37); time (10-120 min). The reaction also was in peg4000 and without it. Checked a restriction enzyme activity.
Still, no result. May be the buffer?
@ The buffer is bought from ThermoFisher - T4 ligation buffer x10. It seems like we observe intermolecular reactions not intramolecular.
I don't see an excess amount of sodium ions, that can be the cause of it - water is deionized. May be the elution buffer is a cause if the reaction on the column goes as the anion exchange...
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