Dear Colleagues,
Can you check my gel result? It is about double digestion of plasmid. But some fragments are seen bad can you say something about that ? Are they contaminated ? or were enzymes cut unspecifically? Do you continue further like transfection using these plasmids? can you trust them? Please help me.
Check attachment "example helps.pptx"
Best
Suleyman
Dear Suleyman , what is the concentration of enzymes used ? and what about the PCR results?
The multiple banding pattern in the cut reaction may be due to star activity of high enzyme conc. in the digestion reaction , try to optimize the enzyme conc and volume in the reaction
I also recommend not to proceed in downstream experiments before checking successful cloning.
Good Luck
If you see your gel carefully each and every lane (cut or uncut, or empty vector) shows this fuzzy smear. did you do RNase treatment after the plasmid prep? If not then I am guessing it is the RNA. Wherever you have high conc. of plasmid, the lower fuzzy band is prominent. I have attached the file for you reference
I agree with Savita and Mohamed. However, I would proceed with cloning if the bands show the expected size - you finally sequence the resulting clones and see whether cloning was successful. By the way, you should check single digest in order to make sure that each enzyme is a single-cutter.
First of all, Thank you very much for your answers.
Dear @Mohamed Rasheed Gadalla,
I used two enzymes; nearly 0.6 ul was used for one them and 1.5 ul was used another one. This is not PCR results. I am undergrad and I am continueing someones' experiment. I transformed them into Ecoli DH5alpha to produce more stock for transfection into S2 cell line. Therefore I digested expression vector to control the insert. I used TANGo 2x buffer for SACI and BAMHI. The table did not show any star activity. Besides one enzyme consentration was less then the actual value to prevent loss of enzyme.
Dear @Savita Shah,
I didnt do RNase treatment besides i didnt know whether it is essential. Thank you for reference file, i can see the smears better there but in Cut A sample which was also isolated with same kit, no smear is seen. I will try to do RNase treatment but is there any other contamination could be ?
Dear @Jochen C Meier,
I dont know now whether i can seqeunce my samples or not but i used old samples which were stocks and i think they confirmed before. I acknowledged that each enzyme cut at one side. But i am not totally sure.
Again thank you very much.
Sincerely
Süleyman
- Badges
- Science method