Is it only one band, 2.2 kB (AOX1 gene)?
Or 2 bands, 2.2 kB (AOX1 gene) and PCR product (588 bp).
I run GS115 with pPICZ (Control given by Invitrogen) but no band was observed.
I only get a band ( more than 250 but less than 500 bp) which i expect should be ghost bands...
The method shouldn't be the issue since my transformants (Mut +) gave 2 bands:
2.2 kB (AOX1 gene) and ~3.0 kB (my insert, 2.2 kB + PCR product, 588 bp).
Hi, Sorry I am bit busy now . so could not ans in detail. Please go through the link below:
http://tools.lifetechnologies.com/content/sfs/manuals/easyselect_man.pdf
I am also attaching a pic which has ans to your ques. Thanks!! Hope it helps .
Tq Gopal. I do have that pdf. But still wonder..
Should it be 2 bands (2,2kB for AOX1 gene and 588 bp for PCR product).
Or only 1 band... Hope to hear detail answer from you soon!
Hi,
Yes if you are using both forward and reverse AOX primers you should get two bands.2 bands: one corresponding to AOX gene and one corresponding to heterologous gene
If you want to avoid any confusion you could also try using gene specific forward primer and 3'AOX reverse primer for you PCR reaction, In such case you should get a single band of 235bp+ your gene size.
Also advantage of having gene specific forward primer is you can be very sure your gene is intact in yeast chromosome. Hope it helps. Thanks
Hello Gopal could you give me information about why I see two bands when I do CPR, what it is the reason for this happen?
Hi, what is the posibillities if i don't see any AOX1 band (that should be 2,2 kb) but i see 2 faint bands that are maybe my target gene and contaminant or primer dimer?
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