What are the primers to get whole length of the sequence?

Dear all Please if someone can guide me.. I have cloned a gene of 1500 bp pairs in FAST-Plasmid. I used reverse primers to sequence the gene. The company sent me 900bp length.. I want to sequence...

11 December 2015 8,729 1 View

Anyone familiar with restriction double digestion of p35S-FAST Plasmid?

10 November 2015 7,135 2 View

Restriction double digestion of p35S-FAST Plasmid..?

Hello every one I want to double digest p35S-FAST Plasmid with Kpn1 and BamH1 1X tango buffer.. The "Double Digest Calculator-Thermo Scientific" recommends the following condition. 1X Tango...

10 November 2015 4,454 0 View

Which (forward or reverse, or both) primers should I use for cDNA synthesis from gene specific primers?

I have extracted RNA from plant immature fruit. I want to synthesize its cDNA by gene specific primers. Please guide me on which (forward or reverse or both) primers are used in such cases.

03 April 2014 9,362 6 View

Can you help me achieve adequate amplification of required band?

I am using 0.2mM dNTPs,40pm primers,0.6microlitre Taq,2 microlitre MgCl2(2.5mM) in a total 20 microlitre reaction volume for PCR. I have kept the temperature the same so far. I need a band size of...

02 March 2014 1,460 13 View

Would the process of extracting RNA from plants without liquid nitrogen be successful?

Would the process of extracting RNA from plants without liquid nitrogen be successful?

02 March 2014 4,430 9 View

Would RNA extraction from plants be successful if i do not use Liquid nitrogen?

Thanks in advance

02 March 2014 3,995 1 View

How should my cDNA band look like on gel?

I want to synthesize cDNA from plant RNA. Please tell me that what should the cDNA band look like on gel, so that I can guess that cDNA has successfully synthesized. Please show me some gel...

01 February 2014 887 1 View

Can anyone help with optimizing PCR conditions for my gene of interest?

I made different dilutions of control cDNA and in each case after running its PCR got the band of 496bp .The Gel pic is as below. But when I ran PCR for my gene of interest with same dilutions of...

01 February 2014 652 3 View

Can anyone help with cDNA agarose gel?

I have synthesised cDNA from plant RNA .The gel picture is given below. Please if anyone could tell me that whether the cDNA has synthesised or not? If synthesised, can I use this for Reverse...

01 February 2014 3,377 13 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

Ester cleavage conditions?

Hi all, I need to cleave the ester in Ethyl 2-amino-1H-imidazole-5-carboxylate to make the carboxylic acid and retrieve the product from solution to use in downstream applications of linking to...

01 March 2021 8,766 3 View

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 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

Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...

25 February 2021 5,712 3 View

What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

24 February 2021 8,618 3 View

Recommendations on homogenizers for RNA isolation?

I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the...

24 February 2021 3,623 3 View

RNA-seq DEGs downstream analysis and reporting in literature?

I used EdgeR to get the list of DEGs (differentially expressed genes) from my liver RNA-seq experiment. I have done the gene overrepresentation and GSEA based pathway analysis. I am currently...

23 February 2021 8,969 1 View