Problem with promoter PCR and cloning - (Aug/18/2008 )
I got couple of problems in promoter region cloning. I am trying to clone 3.2kb region of X gene. The problems are
1. Where, as in the amplification i am getting dimmer bands between 2.8-3kb in the gel. But I felt its very difficult to separate both bands from the gel.
2. After cloning i found only 2.8kb region. But in between 450bps missed when compared with database. Based on bioinformatic analysis CpG island located in the missing region.
Here please do help me regarding this ..
Did you try to make a ligation with the crude pCR instead of purifying the bands out of the gel ? You can do the screening of your transformants directly by pCR I guess !
how different are the strenght/intensity of the desired 3.2kb band and the dimer 2.8-3kb band? Could you provide a picture?
what gel concentrations are you using to purify the insert? You can run a physically longer gel and/or a lower density gel to help separation. And you can you also double gel purify your insert to reduced contamination for small band products. But do remember not to over expose the DNA to UV light as UV will damage your DNA.
How many positive colonies did you check? It should be possible to screen several clones to find one which has the intact insert.
And, is there any interesting feature (repeats...) in the missing 450bp?
Hi it is not probable you have gott dimer band about 2?8kb because dimers can by long aroun 50bp, but you shoulld try special PCR polymerase for GC rich DNA regions, Ecoli s.r.o. has like that but I hope you will find it anywhere