cloning problem... - (May/30/2011 )
hello everyone ...
It has been almost 3 months for me trying to subclone a gene from one plasmid to another...
I cut the gene and vector with BamHI and HindIII enzymes.. the vector is 4.5 kb and insert is 700bp.
This cloning is really giving me pains.
I tried everything... prepared the new vector, new insert, new competent cells(electrocompetent cells / chemical com cells), but the results are zero.
The problem is that i dont get the colonies... the vector has got the Hyg resistance gene and I m using 200 microgram/ml or 150 microgram/ml...
can somebody please give me some suggestions...
thanx in advance...
Hi:
I have a question for you, did you check if the ligase is working?...maybe you need to make a control of this, try to recircle some plasmid.
I don't know another way to help you 'cause a 700pb is not a dificult insert.
Hope can help
Cya
Try to use lower concentration of antibiotic, maybe 50 and 100, or even as low as 25. Do a quick kill curve with one transfomration reaction.
I assume you are using the correct controls? Uncut plasmid, cut plasmid, cut plasmid + ligase but no insert. Are they working properly or are they not working either? If some of them aren't working then it might let you narrow down which part of your protocol isn't working.
If you are cutting the gene out of one plasmid and trying to ligate it to another, I assume you are purifying the excised gene after cutting it out the prigional plasmid? OTherwise it could just be re-ligating to the old plasmid again. Have you run it on a gel to check the inset is cut and your enzymes are working?
Sorry for obvious questions but better to discount the basics before trying to work out something more complicated! Could you say what you hae tried so far?
philman on Wed Jun 1 09:15:33 2011 said:
I assume you are using the correct controls? Uncut plasmid, cut plasmid, cut plasmid + ligase but no insert. Are they working properly or are they not working either? If some of them aren't working then it might let you narrow down which part of your protocol isn't working.
If you are cutting the gene out of one plasmid and trying to ligate it to another, I assume you are purifying the excised gene after cutting it out the prigional plasmid? OTherwise it could just be re-ligating to the old plasmid again. Have you run it on a gel to check the inset is cut and your enzymes are working?
Sorry for obvious questions but better to discount the basics before trying to work out something more complicated! Could you say what you hae tried so far?
Thanx for replying...
well I used the positive control(uncut vector), negative control(cut vector+insert without ligase), ligase control(single cut vector+ligase).
they are all working good as expected. yes i gel extract the gene out of one vector and also gel extract the cut vector after giving SAP treatment.
I also get a lot of colonies at conc of 75 hyg but 100 is nice . I use 150 or 200 as these concs worked earlier.
Does anybody here use in fusion PCR cloning kit...
have a nice day...
Hi technip,
Congrats, well i do fusion PCR and it working..i am not joining the genes with pcr fusion kit..you can! but you need to design special primers...the may i know how long the final base pair you estimate after fusion pcr? dont worry it not take many times..