Identifying cDNA insert - Basic help needed (Feb/22/2009 )
I'm very new to this, so apologies for this basic level question.
I've performed 3 digests -1 with EcoRI, a double digest and a third digest using XmnI with three unknown cDNA inserts.
Having carried out electrophoresis and obtaining the nucleotide sequences for each insert, I now need to identify which sample is which.
The vector used is pGEM-T. I've inputted each sequence into a restriction webmapper to find the point(s) at which each restriction enzyme cuts. To calculate the theoretical size of each insert and in turn identify is which, would I need to subtract the value given at the 'cut site' from the total vector size then add the values from the webmapper in turn to see which corresponds to the band sizes on the gel? For example, the size of the vector is 3000bp, the restriction enzyme that cut each insert cuts the vector at value x and the cDNA at points y and/or z.
Would my calculation be: 3000-x+y then 3000-x+z and check which corresponds to the bands on my gel? Or should both y and z correspond to each of the 2 bands per sample?
Any advice is much appreciated -I've probably made this far more complicated than it actually is but it's my first time.
There is a much much easier way:
Sequence the insert using the standard primers (e.g. M13, Sp6/T7) for the plasmid and then run the sequence obtained through Genbank.
As suggested, i would sequence it.