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Identifying unknown cDNA inserts from restriction digests - (Aug/26/2010 )

I've performed 3 digests -1 with EcoRI, another with both Hind3 and Nde1 and a third digest with XmnI with three unknown cDNA inserts -A, B and C. The possible identities of the inserts have been provided.

Having carried out electrophoresis and obtaining the nucleotide sequences for each insert from an online database, I 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?

What I have done:

On my gel each digest gave two bands for each of the three samples. I took the size between the two bands as one fragment then subtracted this from the size of the vector to give the size of the second fragment.
When using the webmapper, only Xmn1 was found. On clicking on it, I'm given 2 cut sites. At this point, I'm unsure about how to use this information to identify which insert is which and I'm unsure of how to take the fact that my vector is circular and the webmapper results are linear, into account.

Any (simple and step-wise) help would be very much appreciated as I've never done this before although I'm sure it's very straightforward.

I've searched the forum and found a very similar question however, I'm not able to use alternative methods (or carry out further lab work) to work out which insert is which.


Sounds like an exam question....not much more to answer here since you already have the sequence.


NemomeN007 on Thu Aug 26 20:58:35 2010 said:

Sounds like an exam question....not much more to answer here since you already have the sequence.

Er, thanks for replying anyway.

It's not an exam question. As I said, I've not done this type of experiment before and it's not an ongoing investigation just a one-off to learn the technique. I just wanted to know if what I'm doing so far is what I should be doing.