Why I did not get good digestion for phage DNA? I need to determine the diversity of phage isolates, and the approximate genome size from the fragments generated from RFLP. My concentration of phage DNA is 338.97 ng/µl & 1.93 at 260/280 nm. I used 1µl EcoR1 (NEB), 1µl (50x) buffer, 5µl DNA and 3µl Mill Q water for 10 µl reaction.
Hi Nattan, your reaction mixture should contain 1uL of 10X buffer for 10 uL total reaction mixture. If you use 1uL of 50X buffer, you should bring the total volume to 50 uL. H2O should be 43 uL in your reaction mixture, You might have to add more DNA in this case.
Hi Nattan, your reaction mixture should contain 1uL of 10X buffer for 10 uL total reaction mixture. If you use 1uL of 50X buffer, you should bring the total volume to 50 uL. H2O should be 43 uL in your reaction mixture, You might have to add more DNA in this case.
Hi Nattan,
From the picture you attached, it looks like that the DNA had been digested. When you digest a genomic DNA sample with a specific restriction enzyme, it usually looks like that--smear. See the attached sample picture of gDNA digestion for Southern (from web). And later after you probe it (doing RFLP), you will see individual bands. But, next time, to make it better, you should prepare 1x buffer the way as Chandra mentioned.
Dear Nattan
as the previous people mentioned you have to certainly adjust your reaction materials to 50 microliter reaction In my inion you have to digest more DNA (in a reaction of at least 20 microliters and then run the whole lot on the gel. This way you will get a better visual result of the digest. And yes if you digest that kind of meterial you will get a smear.
Best wishes,
Nikos
I think that you have to probe two more simple things. First,read manual of enzyme and check it if you can use BSA for improve the activity enzyme and try with the same time and condition. If the results are the same, you can change the incubation time, for this manner you reduce nonspecific cut. Good luck :)
Hi Nattan Do check the buffer and incubation time is optimum for the enzyme. Is there a different buffer that you can use? You can increase DNA conc to 7-10 ul. Definitely increase the reaction volume upto 50ul.
Dr. Chandra Somasundaram is right. You have purchased and used the 50X ECOR1 buffer instead of 10X. However, dilution makes good result better than first attempt. The products are far from the well, better increase the concentration of Agarose and also not necessary while you taking the digested product to cloning/elution for further process. Fig.2 DNA concentration is very little, try increase the DNA concentration also. All the best.
I am grateful to all of you for your support !..
i am getting digestion from phage DNA with small streak,and herewith i have attached gel image below for your kind perusal and please suggest any modifications I can make in my protocol to improve the gel picture and reduce the DNA streaking.
Hi Nattan
That's great progress in the digest! Changing the % of gel, 0.5-1% higher could give a better resolution, esp the lower mol wt bands. Try reduced voltage while running the gel.
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