Cloning

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Questions related to Cloning

AAV Extraction from supernatant ?

I am trying to perform AAV long-read sequencing using the PacBio protocol. The protocol requires 1.5x10e12 Vg input to use 100uL and get 1ug DNA at the end of extraction, in order to get around...

19 October 2023 665 1 View

Should I use the same primer sequence from the literature while ordering primers?

Sorry basic question. I am aware primers should be described in the 5'-3' direction in the literature. When I am ordering primers I have taken from published papers, should I use the same sequence...

19 October 2023 5,501 3 View

Does anyone know a glue for rocks thin section that is transparent in the infrared (1000-4000 cm-1)?

I want to make a thin section of rock transparent to IR. CaF2 slides can be used as substrate for the thin section but I need to find a glue able to whistand the polishing and that is transparent...

17 October 2023 5,134 2 View

If an active site mutant can't produce product but binds substrate is kcat zero?

If an active site mutant knocks out product formation at all excessive concentrations of substrate and at all excessive concentrations enzyme, but substrate binding affinity via anisotropy shows...

17 October 2023 2,029 4 View

Method to Isolate Yeast Nuclei?

Looking for a straightforward approach to isolate nuclei that does not require the use of an ultracentrifuge... we generally work with mitochondria, however, we are interested in examining some...

17 October 2023 9,907 1 View

Does anyone have experience subculturing Calu-3?

I encountered some problems when subcultured Calu-3 cells. During the subculture process, after trypsin is added to T75, and then EMEM medium is added to the 15 tube, do I need to centrifuge the...

17 October 2023 3,107 0 View

For MS media preparation. I prepared 100mg/mL conc of hormones. For 1mg/L, we will be adding 1mL to the media,for 500ml how much ml should get added?

None

17 October 2023 5,911 0 View

Does the electron beam need to be in the center of the screen when aligning the objective aperture?

When aligning the objective aperture on a F30 in diffraction mode, does the electron beam need to be in the center of the screen? For example look at the picture I have shared. The beam is off...

16 October 2023 2,030 2 View

How do different types of parasites interact with each other within the same host?

Are parasites capable of entering into symbiosis with each other? There are multipartite viruses, the genomes of which are distributed over several capsids and For the infection to proceed...

16 October 2023 1,687 1 View

Can paraffin-embedded tissue be used for immunofluorescence staining?

Hi. Most of the tissues I'm using are embedded in paraffin for HE staining. However, I also need to analyze those tissues via immunofluorescence staining. The tissue is very limited. So can I use...

13 October 2023 7,947 5 View

Can someone recommend a far-red NIR calcium indicator (dye or GECI) that works well?

Have always used Green or red but now need something a bit further red.

10 October 2023 1,990 0 View

Anyone working on lncRNAs? If anyone knows about the screening of the lncRNAs using bioinformatics if pathway is already known?

I already know the pathway but want to know the upstream lncRNAs that regulates that pathway using the datasets and bioinformatics.

09 October 2023 2,777 3 View

Real-Time PCR Troubleshooting: Amplicon Identity, Curve Overlap, and Melting Temperature Discrepancies?

I am encountering several issues in my real-time PCR experiments and seeking guidance on troubleshooting: 1. Inconsistent Amplification Curve Overlap: The amplification curves in my real-time...

09 October 2023 4,920 0 View

Could you please share some simple cures or prevention for COVID19, Cold, Flu or Influenza, and possibly Other Viruses, and Cancers?

Dear Researchers: Could you please share some simple cures or prevention for COVID-19, Cold, Flu or Influenza, and possibly Other Viruses, and Cancers? Updates on Oct. 10, 2023: First, many...

07 October 2023 7,197 2 View

Does anyone have a good shRNA transfection protocol o idea?

Hello everyone. I find myself trying to do shRNA transfection on plasmid against KDM4A protein. I have trouble determining which of the three shRNAs used may have the best silencing effect on my...

05 October 2023 722 0 View

How to make Fse1 cut my backbone?

I have a 22kb backbone that needs to use Fse1 (NEB) and Kfl1 (Thermo) to cut to(21+kb and 700+bp bands). Still, the 2 enzymes come from different companies and their buffers are not the...

04 October 2023 2,254 4 View

How to efficiently use ccdB gene for cloning?

Hello everyone, I'm currently facing a challenge in my cloning strategy involving the ccdB gene. Despite ordering a gene block with a J13119 and a standard RBS, I've encountered a lack of...

03 October 2023 4,679 8 View

Which cell line is best to study GABAergic system?

Hello, I hope everyone is doing great. I need help as I am working on the GABAergic system for insomnia and I used SH-SY5Y (human neuroblastoma cell line). Still, these cells are not suitable for...

30 September 2023 9,845 0 View

How to see phosphorylation of p-IRF3 protein in mouse macrophages cell line? I used HMW poly I:C(10ug/ml) to stimulate the cells but no results?

I collected samples at 30min,1 hrs,2 hrs,6hrs,,12hrs and 24 hrs. But still couldn't detect phosphorylation. If anyone has done it, could you please suggest me better stimulation and collection time.

29 September 2023 5,035 2 View

When preparing a mineral salt medium containing glucose, you can add glucose to the salts and sterilize it using an autoclave ?

when add glucose to MSM and sterilization in autoclave ,color of medium become red or brown.

27 September 2023 7,624 2 View

Should DMEM media be supplemented with 10% FBS or not for scratch wound assay to check the effect of treatment on cell migration?

I want to assess the cell migration rate through scratch wound assay for the hydrogel. I have read the literature and in some research articles it is mentioned that DMEM media was not supplemented...

26 September 2023 4,464 9 View

Do you have good references for protein precipitation with PEG?

I am trying to find at least 1 good article that describes clearly the molecular mechanism behind protein precipitation with PEG6000. For some reason I'm not able to find thin information clearly...

26 September 2023 5,051 4 View

I need a detailed protocol for the testing of CD4 and CD8 T cells responses in splenocytes using flow cytometry?

I have an idea about the overview of the test, however, I need step-by-step details to follow in carrying out the test.

26 September 2023 4,070 5 View

I am doing a PCR for a 650 bp DNA for cloning purpose but getting a PCR product of 400 bp even with different conditions. what can be the reason?

1.Non-specific band at 400 bp, even though by gene size is 650 bp 2.No primer dimer 3.PCR Conditions using right now: Denaturation - 98 degrees (45 sec), ext.- 98 degrees (30 sec)...

25 September 2023 6,958 2 View

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