Hi. I have planned to use the plasmid Lenti-multi-guide (Addgene Plasmid #85401). for mig cloning, and BsmBI as restriction enzyme https://international.neb.com/products/r0580-bsmbi#Product%20Information. The Plasmid is a empty vector with the possibility of delivery of multiple sgRNAs.
BsmBI is a Type ISS enzyme so it recognize asymmetric DNA sequences and cleave outside of their recognition sequence. BsmBI has 3 cutting sides within the lenti-multi-guide; 386, 8684, 9233. The oligoes is designed so they can be inserted into each of the individual cutting sides.
I have now performed the digestion 5 times and i have only gotten one positive result (so all cut ) figure added. Sadly I dropped the agarose gel on the floor and I couldn't use it for DNA extraction. I have just redone it and with no positive digestion/very little.
I have tried following for optimization : to use two different BsmBI enzyme stocks, grown new bacterias and redone the miniprep several times. I have used a 1% and 0.9% agarose gel.
The BsmBI manufacturer, biolaps, claim that the restriction should be done at 55 degrees for 5-15 min, but this doesn't work. I am instead incubating at 55 degrees O.N.
I have thought about trying to use a smaller tube, because I see condensation in the 1.5 ml tube and try to increase the plasmid DNA conc.
The lenti-multi-guide plasmid concentration after miniprep is : approx. 60 ng/ul
Have anyone tried to use this set-up and what did you do? Otherwise any suggestions?
I can see several publications have use similar set-up with succes.
Additionally then I am unsure the band around 10.000 kb is the plasmid that have been cut 1 time or if it is the uncut plasmid, and if I just should use this for the cloning.
Please comment for additional information
Thank you :)
sorry, I think the problem is the quality of your plasmid, sometimes, the mini prep plasmid does not work well. you could try midi the plasmid and do the RE.
hi
1- your digestion seems ok
2- you can used from BsmB1fast digest thermo ( 10min , 37c )
http://ulab360.com/files/prod/manuals/201209/13/449401001.pdf
3- try to recovery miniprep plasmid yield l
A: ( E.coli , LB+ Amp 100ug/ml , shaking incubator 250 RPM in Lesley cap tube)
B:you can used from The QIAprep Spin Miniprep Kit
4- Run the plasmid digested in low-melting agarose gel 1% for cloning
good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes