Is it possible to express and purify non-muscle myosin II S1 fragment from bacteria?

One of the proteins in S.pombe that I am working on is tagged with FLAG, so can I still call this strain as wild type as it contains a fragment of foreign DNA? If no, what is the right term for it?

I’d like to use Quantitative Telomerase Detection (QTD) Protocol (Based on QTD Kit by Allied Biotech, Inc. Cat. No. MT3012)

But I Couldn’t find it's company for buying!!

Can you help me please?

Thanks

Hi, i have a gene that i need to subclone in pET28 and the resultant expression constructs used to transform E. coli JM109 (DE3) cells. The resultant colonies will be screened for positive transformants. The plasmid isolated from the positive clones will be used as a template in a PCR aimed at amplifying and fusing the 3’ - end of the toxin-encoding genes to the 6X histidine codons that are part of the pET28a vector.The PCR products will be subcloned into yeast expression vector pKOV96 and the construct used to transform Y. lipolytica.

MY QUESTION IS HOW DO I DESIGN PRIMERS WITH His-tag for the pKOV96 vector? I need to have 2 set of primers (pET28 and pKOV96). The reverse primer for pKOV96 must have a Hi-tag at the C-terminal. 

Anyone who can help?

I generated several transgenic Arabidopsis lines that contain a T-DNA in which two transgenes (Gene A and Gene B: see attached figure for more details) are linked. Each transgene has its own promoter and terminator. When I measured the relative expression of each transgene in the transgene lines, I found that the expression of each transgene was highly variable between lines which is probably due to the integration of the transgenic cassette in different genomic position. Most importantly, there was a significant difference between the expression of Gene A and Gene B in most of the lines whereas equal expression was found in one line. As both gene experience the same position effect,

1. Why was there a difference in expression between Gene A and Gene B. I guess this probably due to the difference in promoter strength. If this is due to promoter strength, then why in one line they are expressing equally?

2. Is there any other explanation besides those I mentioned above?

3. How should I test whether the difference in expression of Gene A and Gene B in the same line is a promoter effect?

Recombination, Plasmids, Electroporation

we are working on Agrobacterium-mediated Transformation in Capsicum annuum,after 3-4 subcultures small shoot buds are initiated but it's not elongated next....

I looked up many protocols for freezing bacteria, but the concentration of glycerol varies from 20% to 80%. How should I choose? Is it v/v or w/w? One of the protocols mentioned spinning the cell first and then adding fresh medium and 20% glycerol.

If liquid nitrogen is unavailable, is it ok to put the mixture from RT into a fridge at -80C? Sometimes cell pellets formed after freezing, how can I avoid pellet formation without using liquid nitrogen?

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updated on April 9

I think I may fine some causes to the previous failure of my recombinant expression E.coli. Some colleagues suggest that there are different treatment for different types bacteria.

Sergii Pochekailov: as for a high-expression-rate strain, it is better to use freshly transformed bacteria, or the expression would drop dramatically.

Jen-Ning Tsai: in the case of the BL21 and its derivatives (commonly used in recombinant protein expression), the manufacturers suggest a lower glycerol concentration for storage of the cells, since glycerol concentrations (> 10%) may lead to plasmid instability.

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updated on June 7

Ignasi Roca mentioned another interesting method for bacteria storage. Using skimmed milk instead of glycerol. Since scraping the vials without completely thawing the sample required a very fast operation, he change into 20% skimmed milk. He get longer survival times, besides, thawing is not an issue anymore! I f you are interested, the protocol is detailed in his answer and attatched paper.

hi, could anyone has his tag and myc tag of nfkb1(p50) or else someone can suggest me name of labs or peoples in your circle if you know someone.

I am genotyping transgenic plants for the presence of transgene which is an artificial miRNA construct. I am using miRNA specific primers to genotype the putative transgenic plants which I recovered in Kan media. Every time I am doing the PCR, I am getting a faint band from DNA of a untransformed plant in addition to some clear and strong band detected in some of the samples (please see the picture). My NTC (water control) is clear, i.e, no band. I used different annealing temperature, various cycles (30-35), different concentration of template but getting the same result. As my NTC is not giving any band, can I take a decision on transgenic plants despite having the faint band in the negative template control? I have attached a gel image where

1-17: Putative transgenic plants

-ve1: Non-template control (water+all reagents)

-ve2: Contaminated DNA of a untransformed plant

-ve3: Uncontaminated DNA of a untransformed plant

ve+: Plasmid DNA

Please give your suggestion on this.

Thanks to everyone