In this pic I want to send no: 1 for sequencing but i think its smear will be a big problem. Please advise me.
What is this? Of course DNA, but what sort of DNA? What is the question you want to answer by sequencing? Please give more details.
If the size is correct, then you can proceed for sequencing after eluting the band as suggested by SY.
The band has lot of background, so be precise with the cutting of the band. Do you send it with your own primes to sequence the PCR product or do you plan to ligate it first to some vector? In the the first case, the background should be very less, in the second case, it does not matter as you will be cloning it and may be send the PCR product or the clone itself. In either case, it will be much neater band than the one you have now.
Your going to get a very noisy sequence result with the sample in lane 1 unless you either clone into a plasmid first, or purify the band from the gel using a kit like qiagen agarose gel purification. Be sure to run the gell quite far first before cutting the band from the gel to get a clean size fractionation first.
For this case, I would suggest to cut out the band, perform gel extraction and possibly run second round PCR, hope for single and sharper band which can be used for sequencing.
I just wonder which primer are you using? You might want to modify your protocol or primers because when I run the 16s rRNA for my bacteria, I don't usually run into this problem.
You can purify remaining pcr product, check it and proceed for sequencing.
You quantitate the gene by nano drop and see that you have 300-400ng/ul of DNA in your sample before sending for sequencing.
all the best
Dear Mushtaq, in my opinion if the size of DNA is your expected one, so you can just cut the gel and pure it by the PCR, Gel purification kit, and then with the primer specific for this region send for sequencing.you should only cut the area with the correct size of band.
Best
You can cut this band if it is of the correct-expected size. But the sequencing depends on the concentration of your purified-DNA that measured by the nano drop. I would recommend you to pre-heat your elution buffer before use, so you could get high concentration of your DNA.
Hi Mushtaq,
Just curious, had you send out this band for sequencing? How did the result turn out? Did you get a good sequencing read? It will be good you can update us, so we can learn from each other.
dears all
i did PCR product purification for sample line 1 and sent it to the sequencing. the result was Exelantee
You meant 'excellent' [or excelente, in Spanish]. Good job, and thanks for updating us.
I mean very very good. i think the smear was nor a real smear and it was because of the gelRed
It is some kind of smear, and probably not due to GelRed (see your marker lane, the last lane, there is no smear). So, you just purified your PCR product without going through 'running the product, cutting the band out and gel-purified the band'?
no. I have purified the PCR product then i did a new PCR with the purified product
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