How to design probe specific hybridize with a specific targeted gene in Southern blot method?

Dear researchers,

I am working with a plant extract at the moment that I have extracted by boiling the plant in water. To my surprise, the protein content of the extract is very low (0,5%). Other researchers who used other extraction techniques had higher percentages. Now I wonder: Is that because of the boiling? Were the proteins denatured and centrifuged out of solution? What protein contents are typical for hot water extracts of plants?

BTW: Having protein in the extract was not the goal, because I am interested in the polysaccharides. Still would be interesting to know why it is so little.

Thanks for reading!

I've using the same samples for the past few months to run westerns. All of a sudden my bands and lanes are disappearing. I use the same method every time. I run my gel at 85V for 30 minutes then 110V for 2 hours. I've ran duplicates and they still look different.

Southern blot hybridization

Hello,

I performed a single digestion on my plasmids (~6.5kb) using BamHI. The total reaction volume was 10µl (containing 0.5µl(10U) BamHI) and the samples were incubated at 37° for 1hr.

After the digestion, I stored the digested samples at -20°C and run the DNA gel electrophoresis on the next day to check the samples. I used 1% agarose gel and 1X TAE buffer ,and I run the gel for 45min at 100V. (The dye was DL5000(6X) FluoroDye, smobio )

My expected result is: only 1 band should appear in lanes containing completely digested sample (~6.5kb but higher position because of its linear form)

However, the result of the gel electrophoresis (as the figure below) showed smear bands and some of the samples seems to be incompletely digested. In one of the digested samples, there are 2 bands on gel, one is obvious at 12kb and another is above 12kb , no 6kb band was shown.

I'm wondering why? Did my samples ligate with each other?

My questions are:

1) What may cause the smear band or the band shifting in gel?

I was thinking about the possibility of BamHI binding to DNA, DNA overloading, wrong RE digestion condition or electrophoresis condition...etc. Should I inactivate the enzyme by heating after digestion or before electrophoresis?

2) Why are my samples incompletely digested?

Did I set the reaction condition wrong?

Sorry for asking lots of question, and thank you so much for answering!

I have to make a screen, based on 1 hybrid assay in bacterial cells. Technically, I just need to co-transform 2 plasmids, the substrate, and the binding protein plasmid and select for the phenotype on the plate with 2 antibiotics.

I worry that I can lose a lot of transformants because they will either the substrate plasmid or the binding protein carrying plasmid. My substrate library contains a high variability of sequences, and to cover it X100 at least, I need to make the co-transformation as efficient as possible.

There is a way to make competent cells with the binding protein plasmid already because it al the same in one transformation reaction, but on the other hand, I have 77 binding proteins to screen, meaning 77 batches of electrocompetent cells. It would be a lot of work, and I would like to know, does it worth it, or not?

So, what would be your advice for the highest efficiency of co-transformation: 1) competent cells with 1 plasmid of interest or 2) to use some tips and co-transform to plasmids with different dilutions?

I need to prepare 100uM solution of acetosyringone for my co cultivation experimentsbut I'm confused in calculation of concentration, can someone please verify my calculations for 100uM acetosyringone? what amount of AS should be used to make 100ml of 100uM AS solution?

For one protein sample, once boiled in laemmli buffer and stored back in -80C fridge, do you still need to reboil it for later use?

On an earlier discussion I have asked for no PCR amplification in my snail DNA. Based on the suggestions by experts I have changed all of my reagents to Taq from Sigma, DNTP mix from Thermo Scientific, and primers from sigma all of them are fresh. highly pure DNA was isolated using Quiagen kit. Again in the PCR  amplification was not observed. Iam using the same conditions for amplification from the beginning and got amplification properly till december last year from then no amplification is observed. Iam using the 16s rRNA gene for my study and also tried a variety of annealing temperatures and many machines but no use. Good amplification was observed earlier at a temperature of 45 degrees. Pleas help me out with valuable suggestions since no amplification is observed even for all fresh reagents.