I am working on identification of copy number in a recombinant Chinese hamster cell line by southern hybridization. I am having issues with the transfer of DNA onto positively charged nylon membrane. As indicated in the gel image, a lot of DNA is still observed in the gel post transfer to the membrane manually. I have loaded around 18-20 ug of genomic DNA in each lane (2, 3, and 5). I have used 10X SSC buffer for the transfer of the DNA, and the transfer time was around 18 hrs. Kindly suggest me ways to improve the transfer efficiency of the DNA onto the membrane, so that there will be good amount of DNA on the membrane to give pick up signal. I have been using DIG labeled probe for detection, I am either getting a very faint signal or no signal. Thank you.
In the early days of southern blotting if we had large dna fragments which migrated slowly through the gel after electrophoresis we soaked the high molecular weight end of the gel in HCl for a few minutes before blotting. Depurination made the large fragments smaller and they transferred out of the gel more easily. You can also get a similar effect by putting the largr dna end of the gel on a trasilluminator to break up the dna. Possibly using a lower percentage ge would help the dna to move from the gel onto the membrane
Paul Rutland Sir, we have already treated the gel with 0.25N HCl for 15 mins for depurinating the DNA fragments in the gel. We have used 0.8% Agarose gel, In spite of that transfer onto membrane is poor. Thank you.
In that case what you have done is correct and lowering the agarose will only make the gel very hard to handle so all I can suggest is to make sure that all of the SSC diffuses through the gel ( no leaky areas allowing the ssc to absorb round the edges of the gel and give the transfer at least overnight and maybe 24 hours to transfer out of the gel Anil Kumar but mainly to make sure that the plastic film masks the edges of your gel so that the ssc must go through the gel into the membrane and paper towels and not round it. Make sure that the ssc amount is enough to transfer overnight and is not running dry after a shorter time
When you load the samples in dense loading dye all of the sample goes to the bottom of the gel and then runs in the lower part of the gel. It may be worth considering a thinner gel (lower volume) and turning the gel upside down so that the transfer membrane is on the lower side of the gel so the dna has less distance to travel when migrating out of the gel
Paul Rutland Sir, we have taken enough care to prevent the short circuit of the buffer as well as maintaining enough buffer in the tank. We are also turning the gel upside down. We have been giving 18-20 hrs for transfer of the DNA on to membrane, as you are suggesting we will allow the transfer to happen for at least 24 hrs. Also, we will reduce the thickness of the gel. Thank you.
Good luck Anil Kumar . I will be interested to know what the solution is when you do get it resolved
if I remember well there is also a NaOH step before transfert. At the end of the transfert the gel is very thin (all the water has been absorbed by the paper towel you put on it. be carefull that the paper towel does not touch the buffer reservoir and taht it does not soak it up directly (not creating a "stream" from the reservoir to the paper towel going trhu the gel...)
Didier Poncet We did use NaoH and NaCl for denaturing the DNA fragments in the gel before transfer. We have taken enough care while setting up the transfer so that there is no direct contact between the paper towels and reservoir buffer. Thank you.
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