Dear all,
I have transformed my Ecoli with a clone of size (14.3kb). Plasmid alone is about 13.9kb and insert of size 432bp. In 1:3 ratio I got around 200 colonies, negative control plate (only cutplasmid and no insert) was empty. In 1:6 vector: insert ratio i got around 40 colonies. I did miniprep of 20 colinies from 1:3 plate and then diagnosis restriction analysis and PCR of the isolated DNA. I did this twice, in PCR , out of 20 samples 4 were positives, i could clearly see my insert near the 500bp band. With double restriction digest I did not succeed to get my insert cut , only I see 1 band of my vector.
Could it be that the R.E are not working (HINDIII and XbaI) or my PCR gives false positives of the same size like my insert.? How can i check this ?
XbaI is blocked by overlapping dam methylation. If the XbaI recognition site is preceded by GA or followed by TC, the Dam methylase within most E. coli cloning strains will methylate the GATC site.
For example, XbaI sites gaTˆCTAGA and TˆCTAGAtc will be blocked by methylation. To avoid dam methylation use a dam-deficient strain such as dam-/dcm- Competent E. coli.
Thanks for the answer. To get this strain may be difficult and time required. Is it common that the PCR control shows two bands of the size of my insert? maybe i should repeat the PCR and do the digestion again, not double digestion but individualy, each R.E seperatelly
You can try use a different set of restriction enzymes that will recognize different regions of your vector and digest it with them and compare if your insert included band has the right size.
Why not just go ahead and sequence 2-3 from the clones that were positive in the PCR reaction? If this is OK, just use if for your experiments. You probably already have it cloned OK...
Thanks Nagarjun,I will try to do the restriction digest with different enzymes.
Mr.Ribert, the problem here is that i need to complete it by the next Monday (so it is time limited), it a part of my Msc. In order to sequence it it would take some days that I dont have unfortunately.
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