I am planning to do Insitu-hybridisation. I have done the cloning finally . I cloned the PCR product in pGEMT easy vector . after doing the miniprep. I did the restriction digestion with sspI.
size of insert is 500 bp,
pGEMT easy vector- 3015
avaII cuts in insert at 11 positions and in vector at 1533,1755.
after running the gel I got the gel bands at 2000,1256,222 . and 1694,1562,222
which ORIENTATION I should consider for probe preparation.? how to do the linearisation? and How to select the T7 or SP6 polymerase for invitro transcription
Thanks
If you are doing in-situ hybridization against the genome (DNA) then it shouldn't matter since your target is double stranded.
If you need a specific orientation then you have not provided enough information for anyone to analyze. You can take the sequence of pGEMT and add your insert into that sequence and create the two possible plasmids with both orientations in silico. Then you can do the restriction maps with whatever enzymes and see which one aligns with what you see.
Since I do not have a map of the construct I can not tell you which is which.
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