Seeking Research Collaboration in Cellular and Molecular Biology

For researchers in molecular biology and genetic engineering, I would like to know the most successful genome sequencing technique(s) currently used worldwide?Thanks in advance.

I have to estimate the growth of my bacterial cultures spectrophotometrically and I read articles of measurement at an OD of 600nm. Also what to do if the values exceed 1. What is the proper method for the measurement of the same.

Hi. I want to synthesize this blaTEM gene. This is the FASTA sequence, but I don't know which part is related to coding sequence, Can anyone help me?

(my major isn't related to microbiology or genetic, so I don't know the exact procedure of genetic basics,)

and one more thing, after synthesis how can I know how many copy numbers are there in my sample to generate qPCR standard curve based on that?

Hello ResearchGate group,

As M.D.-Ph.D. scientist working on molecular biology, I am wondering if anyone is interested in joining efforts for creating PCR standardization statistical tools.

Best regards,

Alexios

I found that fingerprint analysis of oligonucleotide fragments was used in molecular biology in some top-tier journals in the 1970s, but it seems difficult to find such experimental methods in current journals. Why is that? Does this experimental approach have any defects? What methods can be used to replace it now?

Hello,

I am currently working on a cloning a 6 kb insert into 14 kb vector by Gibson Assembly. Following heat shock transformation into chemically competent TOP10 cells, I had multiple positive colonies as identified by colony PCR (1 vector-specific + 1 insert-specific primer).

However, following miniprep all colonies were found to contain the original 16 kb vector (all plasmids were run on a gel as a 6 kb insert difference should be easily distinguishable from the 14 kb vector + two colonies were sequenced by whole plasmid sequencing to verify). All fragments are amplified by PCR, and I use DpnI digest + small template amounts (1 ng) to minimize template carryover. I also use a blank plate which substitutes the insert for an equal volume of water. The blank plate had only two colonies compared to 12 on the transformation plate. This cloning procedure has also worked successfully for me in the past with smaller fragments (<15 kb total construct size).

I am suspecting that the cells were unable to take up the 20 kb plasmid, and colony PCR was detecting the successful Gibson construct which was spread on the plate during transformation. I emailed ThermoFisher and they said 20 kb in TOP10 by heat shock should be fine, but I am wondering if anyone else can share their experiences. If chemically competent TOP10 is not feasible, should I think about electroporation or using an alternative strain?

In recent months, I've been running gels for a fragment around 430 bp on 1.2% agarose gel with a voltage of 120 and a 100 bp DNA ladder, and it has been working without any issues.

After a two-month break, I’ve returned to this routine, but now the runs under the same conditions are turning out as shown in the picture. Does anyone have suggestions on how to improve this? I don’t think it’s an issue with my samples, as even the DNA ladder itself is showing problems.

Dmt protection is a important step allowing protection of the hydroxyl group in the 5'OH in the nucleotides. This 5'OH specific protection allow a sequential coupling of the nucleotides, forming a obligonucleotide.

As we know DMT protection is follow the sn1 reaction manner. And for sn1 reaction, primary alcohol is less reactive than the secondary alcohol. Therefore, beside steric hindrance I would like to know why DMT is 5'specific. Thank you so much