Help with Purifying DNA Hairpin - (Feb/03/2009 )
I am using 5'-end labeled DNA to study the cleavage patterns of bleomycin analogues on a 16 nucleotide DNA hairpin. After labeling the hairpin I run the sample on a 20% polyacrylamide gel and cut the hairpin out of the gel.
Using the crush and soak method, I purify the DNA and run the cleavage assay. We then run another polyacrylamide gel to determine where the bleomycin analogues have cleaved the hairpin.
I continually get the same banding pattern across all lanes of my gel...even the control lane which contains only my hairpin DNA (only one band should be visible...there are 4).
I do not know if the sample is impure; this wouldn't make sense since I cut out only one band from the acrylamide gel after labeling the hairpin.
Unfortunately, I know very little about the hairpin that I am using. It was synthesized before I started in this lab and the researchers who used it previously have left the lab. Another downfall is that we do not have a formal biochemist working in our lab...most of our researchers are organic chemists so I do not have anyone to turn to for advice with this issue.
Any suggestions as to how to proceed with this assay would be greatly appreciated.
rae.giro on Feb 4 2009, 02:26 AM said:
Using the crush and soak method, I purify the DNA and run the cleavage assay. We then run another polyacrylamide gel to determine where the bleomycin analogues have cleaved the hairpin.
I continually get the same banding pattern across all lanes of my gel...even the control lane which contains only my hairpin DNA (only one band should be visible...there are 4).
I do not know if the sample is impure; this wouldn't make sense since I cut out only one band from the acrylamide gel after labeling the hairpin.
Unfortunately, I know very little about the hairpin that I am using. It was synthesized before I started in this lab and the researchers who used it previously have left the lab. Another downfall is that we do not have a formal biochemist working in our lab...most of our researchers are organic chemists so I do not have anyone to turn to for advice with this issue.
Any suggestions as to how to proceed with this assay would be greatly appreciated.
preeti_RNA on Jul 8 2009, 02:37 PM said:
rae.giro on Feb 4 2009, 02:26 AM said:
Using the crush and soak method, I purify the DNA and run the cleavage assay. We then run another polyacrylamide gel to determine where the bleomycin analogues have cleaved the hairpin.
I continually get the same banding pattern across all lanes of my gel...even the control lane which contains only my hairpin DNA (only one band should be visible...there are 4).
I do not know if the sample is impure; this wouldn't make sense since I cut out only one band from the acrylamide gel after labeling the hairpin.
Unfortunately, I know very little about the hairpin that I am using. It was synthesized before I started in this lab and the researchers who used it previously have left the lab. Another downfall is that we do not have a formal biochemist working in our lab...most of our researchers are organic chemists so I do not have anyone to turn to for advice with this issue.
Any suggestions as to how to proceed with this assay would be greatly appreciated.
Hi,
have u checked the DNA after elution on the PA gel, try loading one aliquot without cleavage reaction buffer....n see if its intact or not.... i would suggest to load the buffers without DNA as well to check if everything is free of any external contamination. what protocol you use for soaking the DNA after crushing the gel and under what conditions you elute them....if its some buffer or column. you can cross-check this also by loading the gel piece before going for gel elution to rule out any possibilty of degradation during elution process..i hope this can help
