Hello everyone,
I hope you are all doing well. I would like to kindly ask for your advice regarding my restriction digestion electrophoresis results.
My target gene is 5385 bp, with the vector being 3980 bp and the insert 1385 bp. There’s an additional band near the top of the gel, which I suspect may indicate incomplete digestion. However, I have already carried out the reaction for 3 hours. Could you please advise if there are any ways to further improve the digestion efficiency?
Here are my reaction components:
- Plasmid: 5 µL (320 ng/µL)
- Enzyme 1: 0.5 µL
- Enzyme 2: 0.5 µL
- Buffer: 1 µL
- BSA: 1 µL
- ddH₂O: 3 µL
Thank you very much for your help and suggestions!
I think that you are correct in thinking the large band is due to partial digest. If both of these enzymes cut at 37c then do the digest overnight.It will not do any harm or cause star activity but this band is far away from the insert band so if you want the insert for the next experiment you do have plenty of insert to work with
you can improve your digestion by increasing the final volume of digestion;
- do not exeed 10% final concentration of glycerol (enzymes are in 50% glycerol)
- DNA concentration at 0.1ug/ul final concentration
- check if your enzymes are efficient in the same buffer (maybe you can start digestion with the one that requires less salt then do the second digest after adding salt concentration for the second enzymes ... (or use enzymes from the "one buffer for all" supplier )
... why did you put BSA??
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