No band after Double digestion - (Apr/01/2008 )
I am running my ligation experiment for the first time.
i had extracted my plasmid with Qiagen spin column and i have a very good band on the gel (3 band as its circular).
Regarding the insert, i had amplified the insert then purified it and i used the smaller (30 ul) for elution to have a more concentarted one.(its gives me a very clear band too).
But After i did the double digestion for both the vector and the insert i got no bands on the gel.
i am using EcorI and SnabI and i am using the recommended buffer from the company (fermentas) which is BamhIII..
Please i need your Help 
Your gel was completely blank?
I've never used Fermentas (I use NEB), but if the conditions were right and your sequence information is correct, the digest should not have been a problem. It may have been a problem with your gel visualization. Are you using EtBr? did you load the entire reactions on the gel?
I've never used Fermentas (I use NEB), but if the conditions were right and your sequence information is correct, the digest should not have been a problem. It may have been a problem with your gel visualization. Are you using EtBr? did you load the entire reactions on the gel?
Thanks MolBioGirl
i use many controls on the gel like the circular plasmid and the PCR product before digestion and both were clear on the gel. Although i will try the NEB enzymes, i know that they are better than fermentas.
Anyone has other suggestions.
It seems to me either you do not have enough DNA samples at first or just star activity perhaps? How long do you incubate the digestion? and how much enzyme have you add inside?
Thanks
finally i had got the transformant. and as you said ...i have inncreased the DNA concentration.