Failed enzyme digestion with RE in RFLP?

Hi, I have a question regarding my PCR products. Okay so i did some primer optimization for primer 1 using gradient temperature starting 55-60’C and i chose temperature 56.6’C as my temperature as it show a clear band. However, when i did amplification using primer 1 at 65.5’C for all my 60 samples, they showed double bands which are quite questionable as they did not even showed up when i did my optimization. Can someone help me to identify the problem? I did the same step with the same primer and the same machine. Here, i attached my result. For your information, 1e is the band for 56.6’C.

I regularly have low DNA concentrations on the vetigastropod tissues I extract. I have tried fresh tissue and older/museum tissues. We use the Thermo Scientific GeneJET Genomic DNA Purification Kit.

Heating the elution buffer and using less buffer to elute does help, but concentrations are still quite low. Downstream applications are PCR and sequencing.

Super desperate and would appreciate any suggestions on how to increase the yield from extractions!!

Hey all

I added 10uM of primer instead of 0.5uM in a 10uL PCR reaction. What are the expected results? Will the amplification of the desired product happen by any chance?

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.

I am having a problem with the reproducibility of the endogenous controls of my samples in qPCR. I have tested several concentrations of cDNA with the objective of discharging that this was the problem and I have found that for all concentrations the amplification cycle is similar, which leads me to think that I have the reaction saturated.

At this point, should I decrease the amount of syber or primers or both?

I am using PCR Instrument called SLAN from HONGSHI. Any clue?

I have a problem with my PCR amplicon size. The desired size is 758 bp, while I am getting a band size of 1200 bp. No amplifications in the negative control. But in each case, I am getting a band size of 1200bp. What will be the logic behind this?

I have been trying to PCR amplify an ~3kb fragment from cDNA of THP1 macrophage to put restriction sites at both the ends so that I can use it for restriction cloning. My primers are 36bp long with 24 annealed bases, 6bp of the RD site, and 6bp for the enzyme to sit; they have a Tm of ~53°C with a GC content of 44.4% and 41.7%. I set up a gradient PCR from 47C-59C and didn't get a single band of good in any of the lanes (image attached). I eluted my specific 3kb band (gave an inferior yield of 4-8ng/ul) and used it as a template to put another PCR, expecting to see just one band (since, now, only my desired template should've been there taking isoforms also out) but still saw a similar pattern (as seen in the attached image) of smaller non-specific amplicons.

What can I do to have no non-specific amplicons either (so that I can proceed with PCR cleanup instead of gel elution to prevent yield loss) or increase the intensity of my specific band?