HELP!!! - (Apr/13/2011 )
I am struggling with cloning my gene in pET28 vector using XbaI and BlpI as cloning sites since 1 and a half month.
The problem is that iam getting colonies in negative control (double digest with ligase but no insert)as well as in vector+insert. The ratio of colonies in both the cases is 1:1 so it is difficult to screen out. I had also used CIP before ligation (from fermantas)to prevent self ligation. I also checked my enzymes for their activity on gel and both enzyme seem to be working by giving linear DNA. Can anybody suggest anything......
First thing i would do is to check the competent cells, are they free of contamination????and how efficient they are for ligation transformation..
then i feel one of your enzyme doesnt work!!!????, anyhow try digesting your insert and vector by sequential digestion, though this is not necessary for all enzymes at times this works....
then i would completely change the vector to a newly prepared plasmid. then if possible try to change the restriction enzyme i am ok with Xba but no idea about Blp, most important go back to the primer sequence of your insert and check whether the restriction sites are correct and also you added few more bases at the 5' end as per the NEBs suggestion... if you still couldnt pass get back and post your full procedure, may be it will be helpful for the experts here to give you some more suggestions..., good luck....
Gnana....
GNANA on Wed Apr 13 20:01:08 2011 said:
First thing i would do is to check the competent cells, are they free of contamination????and how efficient they are for ligation transformation..
then i feel one of your enzyme doesnt work!!!????, anyhow try digesting your insert and vector by sequential digestion, though this is not necessary for all enzymes at times this works....
then i would completely change the vector to a newly prepared plasmid. then if possible try to change the restriction enzyme i am ok with Xba but no idea about Blp, most important go back to the primer sequence of your insert and check whether the restriction sites are correct and also you added few more bases at the 5' end as per the NEBs suggestion... if you still couldnt pass get back and post your full procedure, may be it will be helpful for the experts here to give you some more suggestions..., good luck....
Gnana....
Hi Gnana
I have already checked the competent cells and they are free of contamination. For transformation efficiency i have transformed 100pg of circular vector which gave 110 colonies. Apart from this i always transform circular vector as a control and shows very good efficiency. Have tried with sequential digestion also but got same results. Enzymes seem to be working as we have individually checked both for their activity. Have tried with new stock of vector also but does not reflect any change in results. However, when i run the vector on gel it gives one band (miniprep kit axygen)and the molecular weight is pretty high(more than 10kb)but the same when linearized gives single band at the appropriate size (5369bp size of pET vector).
I am continously getting colonies in double digest transformants and vector + insert. The ratio of colonies is same in both. Can anybody suggest, should i go for colony confirmation of vector+insert which although can be a waste full exercise.
I am using codon optimised synthesized gene.
You can design primers to your vector that amplify the vector, leaving any desired RE sites at each end (don't forget adequate 5' overlaps). Digest with your two enzymes, but also add DpnI, which will digest the template plasmid. The result is linear DNA with the ends you want, minus any transformable plasmid. This dramatically reduces background from uncut or partially cut plasmid DNA. You can go further by gel purifying the result, but it is seldom necessary.