Problem in cloning with electroporation. - (Apr/16/2007 )
I am trying to clone a M2 lamda light chain DNA (680 bp) into M1g1 expression vector (13200bp).
The vector is a expression vector which contains HCMV promoter, Glutamine synthetase and a Ig heavy chain. The vector is (13) kb long and confers (ampicillin) resistance.
I'm preparing the vector for ligation in this way: Vector is digested with Xba 1 and Sac 1
I checked my prepared vector on gel (before ligation, after digestions) and I do check on gel, got 2 bands: one is about 12 kb, and one is 700bp (after digestion). Then I do gel extraction to collect the 12 kb fragment. Gel check is good.
My insert is (680) bp long and amplified from PCR using primers containing Xba 1, and Sac 1 RE site (with 6 bp from the cut site).
I prepare my insert for ligation this way: insert is digested with same two RE. I did not do gel extraction for the insert.
I checked my insert on gel before ligation and I saw (correct length?) I did not check.
I am ligating (50) ng of vector with (40) ng of insert in a (40) ul reaction, at (20 C) for (10 min).
I am transforming Stratagene Electroten-Blue competent cells by (electroporation). I know these cells are OK because I plated them on LB and saw they grow.
All the cells are plated on LB+amp overnight. I also try plate on LB+carbenicillin.
I am getting the following results:
My ligation yields (NO) colonies. (Used 3:1, 6:1 ratio)
To troubleshoot things myself, I performed the following controls in parallel with my ligation: I transformed with a ligation containing only (50ng) of vector and no insert, and I got (NO) colonies.
I also tried to transform pUC19 vector as a positive control, got no colonies.
I have been doing this cloning for 3 weeks, and every time no colonies on every plate ( digested vector + no insert, vector + insert, and pUC 19 vector).. My co worker did the same thing before a year ago, and she got results and I followed exactly her protocols. My co worker and I do not know what I did wrong. And I am getting frustrated. 
Please help me to troubleshoot, thanks!
dont be frustrated, keep trying,
to me 40 ng of ur insert is too much, and probably those 6bp at the ends of ur insert are not enought for the recognition of the enzimes, beside the recognition sequence maybe u should add extra 4 bp long each side and then digest.
if ur puc19 vector transformation is not working then it is the transformation the one not working,
did u tried heat shock?
maybe you can prepare ur own electrocompetent cells also, its very easy.
the recovery time after eletctroporation is important, beside of working on the cold room
good luck
keep tryin
The vector is a expression vector which contains HCMV promoter, Glutamine synthetase and a Ig heavy chain. The vector is (13) kb long and confers (ampicillin) resistance.
I'm preparing the vector for ligation in this way: Vector is digested with Xba 1 and Sac 1
I checked my prepared vector on gel (before ligation, after digestions) and I do check on gel, got 2 bands: one is about 12 kb, and one is 700bp (after digestion). Then I do gel extraction to collect the 12 kb fragment. Gel check is good.
My insert is (680) bp long and amplified from PCR using primers containing Xba 1, and Sac 1 RE site (with 6 bp from the cut site).
I prepare my insert for ligation this way: insert is digested with same two RE. I did not do gel extraction for the insert.
I checked my insert on gel before ligation and I saw (correct length?) I did not check.
I am ligating (50) ng of vector with (40) ng of insert in a (40) ul reaction, at (20 C) for (10 min).
I am transforming Stratagene Electroten-Blue competent cells by (electroporation). I know these cells are OK because I plated them on LB and saw they grow.
All the cells are plated on LB+amp overnight. I also try plate on LB+carbenicillin.
I am getting the following results:
My ligation yields (NO) colonies. (Used 3:1, 6:1 ratio)
To troubleshoot things myself, I performed the following controls in parallel with my ligation: I transformed with a ligation containing only (50ng) of vector and no insert, and I got (NO) colonies.
I also tried to transform pUC19 vector as a positive control, got no colonies.
I have been doing this cloning for 3 weeks, and every time no colonies on every plate ( digested vector + no insert, vector + insert, and pUC 19 vector).. My co worker did the same thing before a year ago, and she got results and I followed exactly her protocols. My co worker and I do not know what I did wrong. And I am getting frustrated.
Please help me to troubleshoot, thanks!Until your pUC19 control transformation works, it is futile to try ligations. Concentrate on this problem. Something you are doing is wrong -- type of cuvette, voltage, arcing of the electroporator, quality of the competent cells, SOC addition, growth for recovery. Talk to someone else who does this in your lab (or a different lab) and watch them do it.
My coworker and I tried another ligation reaction with the same amount of insert and vector, but used a new T4 buffer. (we thought buffer maybe a problem in ligation). we transormed ligation rxn, two positive control with pUC19. next day, we saw growth in control plates, but not ligation plates. this proved that the cell is competent and electroporation was good, so the problem should be at the ligation steps, right? any suggestion in ligation step? thanks
How much pUC19 DNA did you transform? How many colonies resulted?