RFLP problem - (Sep/18/2013 )

Hello. I´m working with a 64bp segment form exon 7 of AB0 blood group gene. 

I need to digest the amplification products with enzimes MnlI and NlaIII (Fermentas) to determine genotypes. The problem is that I've tried several digest protocols (the ones suggested by the fabricants, and the ones in Hummel et al 2002 from which we got the techinque, among other variations) but the digestion didn´t take place in any case.

Maybe the problem is that the segment is too short for the enzyme to work?

Has anyone experienced any similar problem?   

I´ll apreciate suggestions, comments, etc. Thank you!

Patricia

What is expected distance between two RE? Did you tried to cut with each RE separately?

Inbox on Wed Sep 18 18:49:23 2013 said:

What is expected distance between two RE? Did you tried to cut with each RE separately?

I always use them separately! 

The expected products are:41/23 with MnlI and 37/27 bp with NlaIII

You may want to check whether your RE's are working. Usually for making primers we add 4-5 nucleotides to allow attachment of RE to site. Your RE site is in middle, I guess it should cut.

Paty on Wed Sep 18 21:03:43 2013 said:

 

Inbox on Wed Sep 18 18:49:23 2013 said:

What is expected distance between two RE? Did you tried to cut with each RE separately?

I always use them separately! 

 

The expected products are:41/23 with MnlI and 37/27 bp with NlaIII

 

how do you visualise the products? If you use agarose gels even with higher percentages it will be difficult to see both products and they might look like one still. Long gels and polyacrylamid gels with appropriate concentration are essential here...

hobglobin on Thu Sep 19 17:36:59 2013 said:

 

Paty on Wed Sep 18 21:03:43 2013 said:

 

Inbox on Wed Sep 18 18:49:23 2013 said:

What is expected distance between two RE? Did you tried to cut with each RE separately?

I always use them separately! 

 

The expected products are:41/23 with MnlI and 37/27 bp with NlaIII

 

how do you visualise the products? If you use agarose gels even with higher percentages it will be difficult to see both products and they might look like one still. Long gels and polyacrylamid gels with appropriate concentration are essential here...

 

I use PAGE 10% 19:1. The visualization is not a problem. I'm using Lambda digested with Mae I wich has a 32bp band and It looks fine, with nice separation from 50bp band (ladder). 

Inbox on Thu Sep 19 17:00:18 2013 said:

You may want to check whether your RE's are working. Usually for making primers we add 4-5 nucleotides to allow attachment of RE to site. Your RE site is in middle, I guess it should cut.

Today I checked with a digestion of Lambda with my Re's and they worked ok, so they are fine. I just can't get digestion products form the sequence of interest (and yes, I have positive controls in case you are wondering)

I'm totally stuck here!