How important is flash freezing for electrocompetent cells?

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How can I transform 50 kb plasmid into ecoli?

I must put 50 kb plasmid into ecoli strain. I think 50 kb is too big for heat shock with ecoli. Is that correct? Will electroporation work? Or do I need to use cos sites and lambda phage...

22 January 2021 8,612 3 View

Bacteria strain received has fungal contamination -- how remove?

I received several strains of bacteria from another lab far away. However, one of the samples appears to have high fungal contamination. When I grow it in broth, I see lots of fungal...

03 January 2021 2,749 16 View

New knockout bacterial strain: Can't electroporate it?

I made a new bacterial strain with an unmarked deletion using 2 step homologous recombination (with sacB counterselection). The full protocol is given in the link at the bottom. The protocol...

01 January 2021 1,580 4 View

Western blot: what causes patchy, uneven background?

Sometimes my western blots have very uniform/even background, other times they're very uneven and patchy like in the attached image. Why is this? I thought my blots were drying out during...

05 December 2020 8,656 5 View

Licor Intercept Blocking Buffer -- sterility and reusing antibody dilutions?

In the past, I've always used Licor's old blocking buffer that included sodium azide. I would dilute my antibodies in the buffer, stain, re-collect the dilution, and re-use it a few times. The...

28 November 2020 5,795 3 View

What mutations in oriE increase the plasmid copy number?

I have a low copy plasmid (ori=pBR322) and I wanted change it to high copy (like ori=pUC). This paper seemed to say that only one mutation was required to increase the copy number: High copy...

13 November 2020 7,292 4 View

Western blot: adjusting volume of buffer for blocking/staining?

For western blots, I normally stain with 10 mL buffer with 1:1,000 dilution of my specific antibody. Recently, there is an equipment change in my lab and 10 mL isn't enough volume to cover the...

27 October 2020 2,749 7 View

Is this correct molecular docking?

Hello, buds I need your help to interpret the attached data. R group in serine is CH2OH, but the data show the two O in the group with hydrogen bonds with a molecule. Is this correct?...

02 October 2020 7,787 6 View

Concentrating protein: comparison of methods?

I must concentrate and purify a lot of dirty lysate and secreted protein samples. I just want to run them on protein gels for western blots. I'm trying to decide the best method of...

10 September 2020 2,911 8 View

Oxygen detection using Ru(bpy)?

I am trying to detect if my nanoparticles generate oxygen. Is there a way I can use Ru(bpy) and obtain an emission spectra? (Any other "quick and fast" method I can use to detect oxygen generation?)

26 August 2020 1,942 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

How can you bring two structures together to create an adhesive bond in LAMMPS?

I have created an Ice 1h crystalline structure and an Aluminium substrate structure and equilibrated both at 250K. Now I need to bring them together in a way that an adhesive bond is created....

01 March 2021 3,325 2 View

Necessary tube lenght to cool Natural gas in a dry-ice cooling bath?

I have a tank which contains natural gas at 250 Bar and 293 Kelvin. I want to cool the gas in a dry-ice cooling bath to about 195 Kelvin. The gas will flow in a copper-tube through the bath with a...

27 February 2021 8,128 1 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View

Dealing with BMDM seeding and RNA extraction ?

biomedical master student here , i am dealing with mice BMDM and would like some help , i seed 0.5 x 10^6 cells/per well in a 6 well plate but when i extract the RNA i get a very low yeild , i...

15 February 2021 1,102 5 View

How to raise temperature in small increments during equilibration in LAMMPS?

All, I am trying to equilibrate an Ice 1h crystalline structure to 250K in LAMMPS. When I tried to do this at 250K directly, the structure lost it's crystalline shape and became disordered post...

14 February 2021 5,747 2 View

How long can agarose embedded tissues be kept?

I am embedding PFA fixed tissues in low melting point agarose prior to sectioning and I am wondering how long can these can be kept whilst maintaining tissue integrity and fluorescent signal?...

14 February 2021 6,688 3 View

What is a suitable method to freeze feeders-free, serum free episomal hiPSC?

Hi everyone, I need your professional advice: I started growing episomal iPSC (commercial cell line). I grow them in StemFlex medium. What would you recommend as the most effective freezing...

07 February 2021 8,591 4 View

How to make Fast Green FCF from powder?

Hi everyone We need Fast Green to electroporate plasmid into mice embryos, therefore we simply need it to see some color in the liquid we are electroporating so that electroporation is more...

07 February 2021 1,671 2 View

Freeze/Thaw of LS174t cells?

Hello World, I have been working with LS174t. Complete Growth Media- 10% FBS/EMEM. ATCC says to freeze with 5% DMSO/CGM. when I defrost my vials, they are not attaching to flask. vials put into...

01 February 2021 8,723 2 View

Hanna Alalam

Yes that is fine just keep them on ice while you move then to the -80C.

Jorgaq Pata

Appearently yes, it will be fine to do that, the efficiency can be slightly lower but the bacteria are still transformable. Otherwise, you can use a dry ice methanol bath and after that, just put them on -80°C.

Gen Gi

Does anyone have any data about how much efficiency may be lost by not snap freezing electrocompetent bacteria?