How long I can store DNA that has been digested with restriction enzymes, gel purified and eluted in elution buffer? Will the overhangs be stable (since ss DNA are unstable) after freezing in -20 for long time? Can I use them for ligation??
I am using E.coli BL21 Plyss for a recombinant protein production. The cells debris becomes too sticky after lysis. I spin down the pellet at 13000 g (that is the maximum available here). The...
07 August 2019 3,689 7 View
The acid base titration standards are working fine. I just want to know if there are any protein-ligand standards. Thanks in advance
04 May 2019 2,208 3 View
I use PCR cleanup kit from Thermoscientific. The extraction of restricted plasmid from gel failed twice but the extraction of insert which was run parallely gave me DNA with good concentration and...
01 February 2019 2,082 18 View
Can I connect the column to a peristaltic pump operated at a low flow rate and leave the other end open to elute the contaminants out of the column? Or will it disrupt the column packing??
01 February 2019 9,855 6 View
I need a huge concentration of restricted product. So can i just digest them in a 50 uL reaction with high volume of enzyme (say 5-7uL). This is just to run the digested product in a single gel...
01 February 2019 2,170 13 View
The crucial points that need to be considered to avoid failures in cloning.
31 December 2018 10,093 2 View
What is the acceptable range when checking the primers using Primer-BLAST
31 December 2018 7,266 4 View
The HiPrep 16/60 Sephacryl S-100 column that we use is clogged with some dark coloured particles at the very top. I tried cleaning up the column with 0.5M NaOH followed by distilled water. This...
31 December 2018 5,567 9 View
Should I add oligos based on the chart flanking both the ends of the restriction site or adding oligos to the end of DNA is enough?
31 December 2018 2,753 6 View
I have purified my PCR product with PCR purification kit by thermo scientific. When analysing the sample in nanodrop after purification I get a very high absorbance at 230 nm. Will this interfere...
11 December 2018 9,487 8 View
I have reverse sequences (AB1 format), can I base on reverse DNA sequences to perform nucleotide alignment, convert nucleotides to amino acids and deposit the sequence in GenBank database?
11 August 2024 5,138 1 View
I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...
10 August 2024 7,480 3 View
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...
08 August 2024 4,645 2 View
After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...
08 August 2024 1,668 3 View
I'm trying to find a DNA extraction method for fungi that does not require equipment and heating. Is there anyone who can suggest an alternative option? Thank you
08 August 2024 4,733 2 View
I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...
07 August 2024 7,535 4 View
I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...
06 August 2024 3,130 2 View
After immunohistochemistry of previously fixed in PFA and EtOH and then frozen 20 μm sections of zebrafish brain, DAPI staining is very weak (right) compared to the same sections stained without...
05 August 2024 9,637 2 View
I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...
05 August 2024 9,059 3 View
I am performing ligation of the plasmid and a target gene. The steps I have taken are: 1. Double digestion of the plasmid and target gene 2. Ligation of the plasmid with the target gene 3....
05 August 2024 2,570 3 View