User:Anthony Salvagno/Notebook/Research/2011/04/08/DNA Unzipping Construct Troubleshooting Preplanning

Purpose

The purpose of this troubleshoot is to locate potential issues with my process. Currently unzipping has been more stretching and my PCR product has been smeary. This could be because of poor PCR reaction or it could be because of the end label molecules. So I want to rule out any and all errors.

Primers

Right now for my pALS PCR I have the following primers:

• F50 - biotin labeled; this is the forward primer for the reaction and it is on the end of the BstXI cut site.
• R4500 - dig labeled; this is the first reverse primer but produced two products so we opted for a new reverse primer
• R4000 - dig labeled; to fix the two product issue

I have ordered two new primers:

• F50 unlabeled - to try to make the unzipping construct without the biotin. Perhaps this is messing up the ligation?
• R4000 unlabeled - in conjunction with F50, to test the PCR reaction and see if it removes product smeariness.

Experiments

1. No label PCR: to see if the reaction is smeary by itself, or to see if the labels are causing that outcome
2. PCR with only reverse primer label: to test if the biotin end is messing with the digestion and subsequent ligation