Hi Muhammad,

if you will forgive me, the question sounds slightly confused.

Phosphate is required on the ends of at least one strand of the DNA for ligation to take place, whether sticky or blunt-ended digestion. For many digestions / ligation strategies, removing phosphate is not necessary.

However, when you encounter problems with the vector re-ligating at a much greater efficiency than insert incorporation (single enzyme digest, blunt digests etc), it is at this stage that phosphate removal is needed to prevent vector closure in the absence of insert - with the donor phosphate on the insert being sufficient to promote ligation.

Usually, the vector is treated with phosphatase (variously, calf-intestinal alkaline phosphatase (CIP) or various other shrimp-derived phosphatases such as Antarctic phosphatase or similar) after restriction. I am not aware of other means by which this is achieved - perhaps there are papers from back in the 1970s with chemical alternatives but conventionally CIP (NEB is usually reliable) is the enzyme I use to eliminate this problem.

Please feel free to discuss this here further. Good luck !

@ Robin Dickinson

Thanks for your response i will try to make my question clear.

I have my plasmid cut with BaglII and treated with pyrobest for blunt ends, and its size is 2.4 kb, my insert is 920 bp and has a restriction site for ECORV, i am working for long time and fail again and again, mean no ligation occurred till now, i also try phosphatase, i had tried different concentrations of insert:vector[1:1, 10:1 etc], beside i also get very few colonies when i transfer my ligation reaction to Ecoli.

If possible please guide me and tell me about the concentration of insert and vector in micro liters, i have no idea about pmol etc.

thanks

Hi Muhammad,

thank you for the additional information - it has made analysis of the situation much clearer.

To recapitulate : your vector has been cut with BglII, and blunted using the Pyrobest polymerase enzyme exonuclease activity. The insert is naturally blunt ended with EcoRV sites. Despite trying several conditions, reaction ratios etc., ligation of the desired product does not happen.

Firstly, I should be clear - in my formative days learning to be a Molecular Biologist 20+ years ago, my mentor always impressed on me to avoid blunt cloning where possible, and to find strategies that obviated this such as PCR amplification of the insert to modify the restriction sites to a less risky strategy compatible with the vector. Even if it involved ordering primers and performing the amplification step, the cloning itself is likely to be more reliable.

I had to look up Pyrobest as it is not an enzyme I am familiar with. One critical detail is very important in your case :

See page 2 at the following link :

http://www.takara.co.kr/file/manual/pdf/R005A_e.v050119.pdf

BglII generates 5' overhangs. Pyrobest's data sheet from Takara specifies that it has no exonuclease activity against 5' overhangs and is a 3' exonuclease only. It may therefore be a valid strategy for certain other restriction enzymes that generate 3' overhangs, but for BglII it is probably not ideal.

This may be a significant clue to the problem you are encountering - it appears that you are not ligating into a blunt-ended vector.

If you want to discuss alternative cloning strategies for your insert / plasmid combination I will be happy to suggest possible options if you can give more information about the vector and the insert.

@ Robin Dickinson

Thank you so much sir for such a brief detail related to my topic, if possible can you discuss other strategies which can make my ligation easy for me, as it is a laborious job especially when no results are obtained. instead of pyrobest what can you suggest for such kind of ligation.?

Thanks for your help and suggestions.

Hi Muhammad,

It's no problem - I benefit from the advice of other scientists here for my own questions so I see any assistance I can give as being a way of giving something back to the community.

Are you able to post a map or sequence of the vector and your insert sequences - if you would rather send them to me privately I think you can use the RG messenger function. Then I can think of a strategy and make some suggestions. Best wishes, Robin

@ Robin Dickinson

sir can you send me your email address through which i can send you map etc?

Hi Muhammad, please use :

rdickinson(at)dcbiosciences.com

remove (at) and replace with @ to get the address.