I eluted backbone from pGHC-GFP vector and insert from CryIIAb after restriction with NdeI and XbaI and ligated them for 1 hour with T4 DNA ligase at room temperature for 1 hour, then I transformed Top 10 E.coli competent cells with this ligation but I didn't get any colonies on pGHC-CryIIAB plates and I got upto 100 colonies on positive plates? It never happened to me before, What am I missing? or is there any fault in my protocol? Is my ligation ok or is there fault in competent cells? Need suggestions, please, help me.
It seems your restriction digestion has not worked so well. You can keep ligation for overnight at 4 to 10 degree celcius.
Other thing, your gene may be toxic.
Did you purify your ligation products? Did you use chromatographic methods or ethanol precipitation for purification of your ligation products? did you air dry the DNA plate after ethanol precipitation? Did you quantify your ligation products either using a Nanodrop or an agarose gel?
Which method did you use for transformation? Heat shocking? electroporation? or others?
Are you sure your T4 DNA ligase maintained its activity? What about your T4 DNA ligase buffer?
What about the activity of your restriction enzymes?
Have you done quality control (QC) for your cut products? QC is very critical after each steps prior to proceeding to the next down streaming procedures.
Yes I did purified my ligation products my enzymes worked really well, I got desired bands after restriction with enzymes, my T4 DNA ligase is also up to date and so as the buffer, I used ethanol precipetation method, I quanified my insert and backbone on gel but not on nanodrop, I used heat shock method, Kindly suggest me where is the problem???? I haven't done QC, how to do it, please guide me?
Yes Sir, I have done that E. coli transformation, I am now far ahead of this problem.
- Badges
- Science topic