cloning problem - (Feb/16/2007 )
The size of my DNA fragment is 4.0kb and the vector size is 6.0 kb.After digestion and DNA elution i got band which is of 6 kb for my vector and 4.kb for my insert.But if i do ligation and transformation,there is no colonies. I also did CIP assay to prevent selfligation of vector.There is no problem with competent cells and the antibiotic containing plate,because i got colonies in the control but not for the ligated product.Could you please suggest me some tips.....
Thank you in advance for the slightest hint.
How long have you digested your vectors? Overdigestion of either the vector or the insert could interfere with ligation.
How much DNA was used in ligation and in what ratios? You could try higher ratios like 1:5 or 1:10.
good Luck !!!
In addition to the above questions,
how long, and how much DNA was treated with what quantity of CIP? Over dephosphorylation can and will damage the ends of your vector.
What kind of ligase did you use? How long and at what temperature did you carry out the ligation reaction? You could try ligating at 16 Celsius overnight (using standard T4 ligase buffer)
Do you known if your ligase enzyme and buffer is still in good condition? Both component can rapidly degrade when subjected to repeated freeze thaw cycles.
check thoroughly every single step........from enzyme digestion to ligation and transformation
u may understand from my name that i am also suffering same like u
How much DNA was used in ligation and in what ratios? You could try higher ratios like 1:5 or 1:10.
good Luck !!!
how long, and how much DNA was treated with what quantity of CIP? Over dephosphorylation can and will damage the ends of your vector.
What kind of ligase did you use? How long and at what temperature did you carry out the ligation reaction? You could try ligating at 16 Celsius overnight (using standard T4 ligase buffer)
Do you known if your ligase enzyme and buffer is still in good condition? Both component can rapidly degrade when subjected to repeated freeze thaw cycles.
Hai,
Thank you for your suggestions.I using T4 DNA ligase.As for as ligation is concerned i kept overnight at 16 deg cel.The amount of the CIP enzyme added is 2 microlit.40 micro lit of vector,2 microlit CIP enzyme,10 microlit 10X buffer,48 microlit MQ water,incubated at 37deg for one hr,then gel elution.
Now i am trying my TA cloning but that is also not working.i am not getting any colonies.Please suggest me some ideas to carryout this.
thanks in advance
mala
How much DNA was used in ligation and in what ratios? You could try higher ratios like 1:5 or 1:10.
good Luck !!!
Hai
Thank you for your suggestion.i kept overnight digestion.Before i was using 1:5 ,i increased the concentration to 1:10 but still i failed..
suggest some tips..
thanks!!!!!!!!
u may understand from my name that i am also suffering same like u
Hai,
Thanks for the suggestions...but still i am not getting the result for my experiments.....how about your experiments????wht is the size of your vector and insrert...Even TA cloning is not working for me...
All the best!!!!
i would definitely try to heat inactivate your CIP for 10-15 min at 65C before ligating.
I also dilute it 1:20 in buffer 3 (NEB) and then use only 1ul of this for a 20ul total reaction - at 37 for 30min
- word on the street is antarctic or shrimp alk are better than CIP
Perhaps you can clean up the CIP first before proceeding ligation.
As for ligation, overnight at 10 Celcius is ok as well.
TA cloning works best under kit. Maybe there arent enough vector or insert in the first place. I think I read somewhere that if more than 10ng of ligated product is used for transformation, it will disrupt the transformation efficiencies as well.
Good Luck! 