I'm studying with pGFP vector for cloning. I have already cultured the plasmid and extracted DNA which is almost 300 ng/ ul. And now i'm trying to cut it with restriction enzyme. For the reaction i use 5 ul from my sample ( almost 1500 ul). It works but i will continue to the reaction with 'gel extraction method' that's why i should see a strong band on gel but it is not shining enough on the other hand DNA Ladder has strong bands. So that means there is no problem with gel. But i couldn't see a strong band of my sample why it is not shining enough? What can i do to solve this problem?
You will lost some of your DNA after each digestion and gel extraction. It seems lost too much in your case, check the concentration of your final recovered DNA to see if it matches the gel density. If indeed lost too much , improve your gel extractions.
Yes, it sounds like you are losing DNA somewhere along the process. Is there any way you can try doubling or tripling the amount you use for the restriction digest before running on the gel?
if you are measuring the whole dna of plasmid and a small insert then when you cut and purify the insert if the plasmid was 2kb and the insert 200bases (for instance) then the purified insert will only be 10% of the total DNA so depending on the size of the insert and plasmid you might not expect very much insert. Also short sequences trap less ethidium bromide so may look weaker than the ladder if the insert is very short
Thank you all for your interest.
But i should say that even my "uncut" sample doesn't shine enough too. Both of them (cut and uncut) have the same brightness. I also tried to use both ethidium bromide and SafeView for electrophoresis.
By the way my insert is approximately 1000 bp and the plasmid is 3340 bp.
I just wonder that if it is related to the extraction method? For example alkaline lysis?
Hi Touraj
i didn't run my plasmid for confirmation but i measured it with another nanodrop, it was the same result. Thus, i don't think that our nanodrop has a problem.
can we see a picture of the gel. it is possible that what you are measuring is either bacterial dna or rna if the purification has not worked well so proportionally there is not much plasmid. Is there high or low molecular weight fluorescence showing on your agarose gel?
thanks For the picture Inci. I do not know the size standard but the sizes of the insert and plasmid if that is lanes 2,3 and 4 are look quite similar if the plasmid has been cut but it does not look like 1000 and 3340. There does seem to be some rna or degraded small dna at the bottom of lane 4 but not enough to account for the low intensities.
Sadly I cannot offer any explanation of the low intensities
Can you give a bit more detail on your DNA purification strategy? Are you purifying from an EndA negative strain of E. coli, and if not, do you have a step to eliminate EndA contamination? Did you treat with RNase? If not, likely most of your A280 signal is due to RNA, and the plasmid is simply more dilute than you anticipate. An A280/A260 ratio of 2.0 or higher is a good indication that you have RNA contamination (a good plasmid prep should give a ratio of 1.8 to 1.9 on a Nanodrop).
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