I am doing SSH ( Suppression Subtractive Hybridization ) of drought stressed Pennisettum glaucum .I have obtained 12 contigs and around 34 singletons from some clones which i randomly picked from the forward SSH library of about 280 clones.After sequence annotation of sequenced clones using BLAST X and TBLASTX ( at TIGR and NCBI) i find that most of my clones align to proteins which seem to be constitutive in nature ,such as ribosomal proteins ,Rubiso activase ,CF0 ATP syntetase b chain,putative elongation factors etc.I wonder if i went wrong with my SSH protocol or is it natural for these to be upregulated ...Im planning to go for qRTPCR of a few of these genes but i am doubtful if i should choose them since they are seemingly not induced by stress .I also have some stress related clones such as putative DREB3 and CBS domain containg proteins ....But that is about it when it comes to genes even slightly related ( seemingly) to drought stress.Please tell me why this observation and whether i got the SSH wrong or is it natural for these genes to show up in a SSH library.
I HAVE THREE MAIN QUESTIONS or DOUBTS
what must i do to increase the number of stress related clones in my SSH library ( other than to increase the number of clones which seems obvious)
WHY is there redundancy in SSH library and how to reduce it ???
and Please suggest the fastest method to screen for such drought induced genes from many thousands of such clones in the SSH library as a quick screening to check for redundancy.
Dear James,
1) Higher the Hybridization time lower the number of clones and higher is their reliability.Conversely, lower the hybridization time higher the numebr of unigenes and lower is their reliability.
2) You can either size fractionate your library and clone different sized fractions seperately or seperate your products on an agarose gel and cut, elute and clone different sized fragments seperately. This can reduce preferential ligation of small-sized products and hence the average length of your products will increase. Bright bands in your gel can be eluted and cloned seperately in order to reduce redundancy, because they contain mostly very few unigenes.
3) Even though some people recommend reverse Northern dot blots to screen libraries for reduundancy I personally feel that it is not much reliable. Hence if you can afford sequencing large number of clones that would the easiest way out but you will have to bear with the redundancy.
4) You can randomly select soem clones and do qRTPCR in order to know the percentage of positives.
Best of luck
DIcto Jose
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