NEED HELP-verification of my inserts - (Aug/29/2007 )
Could it be star activities? Which enzymes did you use?
-rabbitrabbit-
QUOTE (kylvalda @ Sep 3 2007, 05:45 AM)
QUOTE (bsengez @ Aug 31 2007, 10:22 AM)
First, my insert is PCR product. After purified it, I have cloned it into pJET1 plasmid and verified it by sequencing. Then, cut and run in agarose gel. Everything was okey. Then, I cloned it into expression vector and choosed the colonies with proper antibiotics. It cannot have any parts from plasmid now, because pJET cannot survive where my expression vector lives. From positive colonies, I carried out colony PCR, it gives good amplification and the band level is okey. Therefore, I isolated it and cut if it is ok. Unfortunately, I have additional bands. I have also control for digestion. Enzymes, buffers and vector work well, there is no problem. Any idea?
might be a stupid question but:
after cutting out of pJET1 did you run a preparative gel and cut out your insert and did gel cleanup to be sure just to clone the insert into expression vector
and not also some by-products from the restriction?
It is not a stupid question, thanks for asking. After cutting out PJET1, I run an agarose gel and cut my insert and also I sequenced it. I'm sure that it is my insert.
-bsengez-
QUOTE (sheha @ Sep 3 2007, 09:40 PM)
can i know wat enzyme u used... how many colonies u get.. whether all colonies got same result after restriction digestion
I'm using XhoI, NotI and MunI enzymes. I got 3 positive colonies and 2 of them are like that. One of them had the bands I want, therefore I keep on with it.
-bsengez-
QUOTE (rabbitrabbit @ Sep 4 2007, 01:35 PM)
Could it be star activities? Which enzymes did you use?
Most probably, it is not due to the star activities of the enzymes. I'm very careful to choose the best buffer for each just for preventing it to happen.
-bsengez-