2.5X amount of oligo-dT in reverse transcription reaction?

03 March 2016 1 7K Report

I accidentally added 2.5X the normal amount of oligoDT primers into my reverse transcription reaction. What are the chances i have screwed up my experiment?

Laurence Stuart Dawkins-Hall

Hi there. Very unlikely that will make any difference. All of these protocols are fairly general and there are nearly always wide limits for reagent concentrations within which you can successfully operate. So don't worry and proceed as normal

Badges
Science method

More Jeff Zaki's questions See All

Why the need to use different minimal media to grow bacteria?

I am trying to identify bacteria on skin that can utilise lactic acid as carbon source. So i intend to swab skin and then grow bacteria on minimal media supplemented with lactic acid. My question...

11 December 2018 3,432 0 View

Cell Biology field: Relative easeness in publishing papers?

Hi, I am hoping to get opinions/input since I am undergrad and I see certain trends that I am highly curious about. I have been doing lab rotation to get an extensive research experience....

09 October 2018 9,760 0 View

What other phohpshorylated components can i look for downstream of activated FAK and P38 MAPK?

I applied some mechanical stresses on cells and discovered that FAK and p38 MAPK are activated by phosphorylation. For the purpose of publishing, i feel that it will be great if i can look for...

03 April 2018 5,791 0 View

Are they any phosphorylation-specific proteins for hypoxia effect on cells?

Im looking to expose cells to hypoxic conditions in minutes (

02 March 2018 5,548 0 View

Hypoxia induced: No HIF-1a overexpression seen at transcript level but seen a lot of hypoxia and HIF-1A -induced genes?

hi i need advise on the matter i performed RNA-seq after exposing cells to mechanical stress. I had designed a device to deliver this stress to cells. Looking at the RNA-seq data, i see some...

02 March 2018 5,300 8 View

Proliferation assay : manual cell counting?

How is proliferation assessed by manual cell counting? I will be happy if anyone can provide protocol on this. I have been told this method is much useful than indirect assays like CFSE but i...

11 December 2017 2,510 0 View

QPCR: analyses involving in-between plates?

hi i am not sure if it is alright to analyse my data this way. So i prepared 3 samples of controls, 3 samples of condition at 1hr exposure, 3 samples of condition at 6hr of exposure. i collected...

07 August 2017 7,743 5 View

Time course gene expression analyses: RNA seq over mass spec?

why when wanting to look at genes that respond at say 1hr (early response), 6hr(intermediate response), 16hr (late response) it is better to look at RNA expression rather than protein? has it got...

07 August 2017 4,608 2 View

Why is RNA expression analysis better than protein for looking time course gene expression analysis?

why is it that people usually look at mrna expression rather than protein expression when conducting a time course gene expression analysis?

06 July 2017 9,379 0 View

RNA expression vs protein expression patterns upon stimulation- are RNAs more dynamic?

Let say if i stimulate a cell at 1 hr and 6hr, the RNA expression upregulated at 1 hr may not be reflected at 6hr, but same protein upregulated at 1 hr will be reflected at 6hr. How do i explain...

06 July 2017 783 0 View

In PRIMER, is it possible to downweight certain taxa in multivariate community datasets to reflect uncertainty around identification??

I have a dataset with about 80 different species. As usual, some species are very easy to identify with certainty whereas others are more difficult, which means that I am less certain of my...

03 March 2021 8,066 4 View

PACYC PCR not working after several attempts. Would anyone have any suggestions?

So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however...

02 March 2021 1,146 2 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

How to calculate the annealing temperature of a primer pair?

I have to amplify a gene and my primers just reached. The Tm for Forward primer is 64.2, and that of reverse primer is 65.5. Can some one suggest how to get the best annealing temperature? Thanks...

01 March 2021 360 7 View

Anybody with an idea on how do design specific primers for detecting tomato resistance genes tm-1, TM-2 and Tm2^2?

I am trying to identify these 3 genes among some tomato cultivar collections and after aligning some sequences from NCBI, I couldn't find unique sequences to target for specific primers. There...

28 February 2021 606 3 View

What is the ideal efficiency for a qPCR?

hello everyone, I need to do standard curves for my qPCR, what is the ideal efficiency range? I tried a primer (Mglu2 receptor) that gave an efficiency of 90.2%. Is it accepted?

28 February 2021 1,254 3 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Calculating volume of DNA required from DNA concentration?

Hi I am a bit confused. They are asking me to find out the volume of DNA required in ul (a total of 30-100 ng for genomic DNA) from the DNA concentration in the nanodrop reading which was 404.8...

26 February 2021 5,029 2 View

Has anyone used the Illumina ITS1 primer sets?

Hi everyone, Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each...

25 February 2021 6,969 3 View

Can I use TCA instead of TFA for protein analyis in RP-HPLC?

I am trying to analyze lipase using the reverse phase-HPLC C18 column. In the literature, 0.1 % TFA was and 60% ACN was used. Can I use TCA in place of TFA? If any other methods are available to...

25 February 2021 2,388 3 View