Has anybody used mdi gel extraction kit (range upto 10KB) for PB4NU RD fragment elution?

08 August 2018 1 10K Report

Hi!

I need help with PB4NU (14KB) restriction enzyme digested fragment elution. I cannot order a kit specially for this but I need to elute it. The problem is I tried using Qiagen and mdi both and none of it solved my problem(range 10 KB). There is no elute after even digestion 3microgram of PB4NU. All I could get is 6ng/uL and I need it for cloning purposes and its not helping.

I am finally going for a manual purification. Any suggestions please?

Thanks

Sakshi

Erman Kocak

I think it would be helpful if you try to separate the digested DNA using a high range DNA ladder and low percentage gel but you should prepare the gel with two combs, ones at the top and the another at the middle of the gel so that you have pockets on the top and middle

after loading and starting the run you should follow the run and take the DNA back from the middle pocket

Badges
Science topic

Similar topics
Gels

More Sakshi Mehta's questions See All

Trouble in amplifying a particular gene?

I have trouble in amplifying a gene. I am continuously trying to find a possible solution. All methods have failed me somehow. I was amplifying gene A using gene specific primers. This gene has a...

04 May 2018 6,382 9 View

How to make sure if your RNA has been converted to cDNA?

I am trying to amplify a gene from cDNA. The gene contains an intron, therefore, I am amplifying it from cDNA. When I amplified it using genomic DNA, it shows the desired band. But when I am...

03 April 2018 2,499 9 View

How to reduce degradation in Agrobacterium RNA ?

I am isolating RNA of Agrobacterium and when I performed it for the first time it showed no degradation and now it is showing degradation. What can be the possible cause? I used Trizol method...

01 February 2018 5,570 6 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

No title

Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View

Anyone that can help with details in phosphopeptides enrichment?

Anyone know the amount of protein before enzymatic digestion for phosphopeptides enrichment of serume sample in Ti-IMAC? Has anyone ever use Ti-IMAC(the material is from Chinese company named...

28 February 2021 7,310 3 View

How can I represent SDS-PAGE results for machine learning analysis?

I am trying to classify and analyze the results of an SDS-PAGE based array for bacterial detection using machine learning, but I have trouble finding the best way to represent the results with...

27 February 2021 9,176 3 View

Why my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 3 View

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 View

EMSA troubleshooting. Protein-DNA complex not migrating, cold probe lane with multiple bands. Can anyone help?

I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA...

24 February 2021 8,325 1 View

Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

I am trying to clone 2 copies of the same mammalian gene into the pSF-CMV-Ub-Puro Ascl (contains HindIII, KpnI and NheI cut sites) plasmid. This is what the final product is supposed to look like...

24 February 2021 6,310 3 View