thank you sir for your reply but i am doing double digestion with EcoR1 and BamH1.
and if their is insertion of multiple inserts so what should I need to do ??
Dear Juhi,
You might want to alter the concentration of insert used. Sometimes to increase chances of getting positive clones we use a higher concentration of insert, but in presence of ligase and other suitable conditions, inserts can be ligated to one another and then to your vector backbone. For eg. RE1 and RE2 are the two restriction enzymes generating sticky ends re1 and re2 respectively. Now, during ligation you may have re1-insert-re2-re2-insert-re1. This might as a whole be ligated to your vector.
But even then it is not possible to distinguish such cases from single insertions in gel electrophoresis after double digestion, where you can only visualise the insert release. Although such concatamers increase the plasmid DNA size and decrease its transformation efficiency. So, it is unusual to get such insert-insert ligations. Even then, if you are getting a higher bp band, after complete double digestion then it is likely not to be multiple insert ligation, as during digestion all inserts will be released and they will migrate as a single band in agarose gel and you won't be able to tell the difference.
So if you are getting a higher band than expected it might be due to some other reason. Another way out is to get the plasmid sequenced and then you can surely know why you were getting the higher bp band.
Hope it helps!
thanx tias saha for yor reply. 😀 i ll try to reduce the concentration of insert and what if i ll treat my insert with cip just to minimize the chance of insert insert ligation (i knw dat os not d case as u said nd i agree wd u but just to eliminate this reason ).
Dear Juhi,
You can use phosphatase and check for this higher bp band. Although phosphatase treatment is generally given to vector to avoid vector vector religation and minimize chances of negative clones, but insert can also be subjected to it. Either one will minimize false positive clones.
All the best!
Juhi, make sure that the cut is complete with both the RE. since they have sticky ends, there might be a possibility that there is end to end ligation as well. What are your conditions for digestion and ligation
Hi Juhi
to be sure your products are digested by both enzymes do sequential digestion. and you can elute the higher insert band and try digesting it again with same enzymes. if it still gives one single band of >1.5 kb size then your insert preparation is probably contaminated. If it gives you the band of 1.5 kb size then it can be a double insert. Run this digestion along with digested vector and digested insert. sometimes linear DNA behaves differently. If it still doesn't work then as Saha said sequencing the band is ultimate solution to be sure of what it is.
All the best
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