I am trying to isolate RNA from THP1 cell line using manually made TRIZOL reagent, I am getting good intact RNA but along with it I am getting huge DNA contamination. So I want to somehow minimise the DNA contamination.
Ai
ni
pH 4.5. Pure phenol. 10 volumes of Trizol to one volume loose packed (70 x g). Shear DNA with 25g needle. Spin 12 x g. Collect 80% of aqueous phase.
I use for the THP-1 either premade TRIZOL or to isolate the RNA the RNA-easy kit from Qiagen.
Here are some interesting patents that may be of use to you. Please, see them.
1. US5346994
2. US4843155
3. US8367817 B2
Out of curiosity, couldn't you just DNAse treat your final isolates?
@John Hildyard i tried many times but RNA is getting degraded after DNase treatment, i am using Thermoscientific DNase 1
See the classic paper by Saatchi and Chomcinski
Hello,
Home-made recipe for 1 L:
Reagents Final Concentration
Phenol in saturated buffer 380 ml 38 %
Guanidine thiocyanate 94.53 g 0.8 M
Ammonium thiocyanate 76.12 g 0.4 M
Sodium acetate, pH 5.0 33.4 ml of 3 M stock 0.1 M
Glycerol 50 ml 5 %
DEPC-Water Adjust the final volume to 1 L
Hope it helps
Regards
I am doing RNA dot blot experiment to probe epitranscriptomic changes in brain tissue. I am not getting good signal with total RNA. I doubt my denaturation step as I am heating my samples at 95...
06 July 2017 9,488 1 View
I have seen many papers performing divergent PCR to detect the circular RNA products. I am not sure if the reverse transcription of circular RNA yields circular DNA with random hexamers.
05 June 2017 9,487 0 View
I am working on H1 human embryonic stem cells and culturing them on matrigel coated plates. On the second day of passage, I could see the media appearing cloudy and plate looks grainy with a lot...
06 July 2015 3,641 8 View
I am working on H1 cells, I am sub-culturing in 10 cm dish coated with matrigel at 1:10 split ratio of confluent cells. I am having this frequent problem of cells not getting colonized on 3rd of...
05 June 2015 6,956 2 View
There are many methods for quantification of gene expression (fold change calculations) through real time PCR such as Pfaffl method, delta ct method, standard curve method. So I want to know which...
07 August 2014 2,192 4 View
I was trying to analyse expression levels of certain genes after treatment of monocytic THP1 cell line with antigenic proteins through qPCR and i tried using the reference genes beta actin and...
07 August 2014 5,082 7 View
mRNA stabilization as a result of translation termination.
10 November 2013 7,788 2 View
I want to know the reason why interactions between translation termination factors and EJCs lead to mRNA decay during NMD.
10 November 2013 6,166 0 View
Utility of real time pcr over northern blotting
10 November 2013 6,204 3 View
I want know exact mechanistic model of EJC, Upf proteins triggering NMD.
09 October 2013 6,739 2 View
Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...
11 August 2024 7,082 3 View
I have reverse sequences (AB1 format), can I base on reverse DNA sequences to perform nucleotide alignment, convert nucleotides to amino acids and deposit the sequence in GenBank database?
11 August 2024 5,138 1 View
I have been doing the m6A dot blot for a while with no improvement, I am extracting the RNA, and I can see the dots although the three biological replicas give a different reading on the memberan...
10 August 2024 8,539 5 View
I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...
09 August 2024 9,621 0 View
Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...
09 August 2024 2,824 3 View
I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method...
08 August 2024 9,882 2 View
After performing symmetric PCR, PCR purification was performed. Afterwards, asymmetric PCR was performed using the PCR purification product as a template, but no ssDNA band was confirmed in the...
08 August 2024 1,668 3 View
I'm trying to find a DNA extraction method for fungi that does not require equipment and heating. Is there anyone who can suggest an alternative option? Thank you
08 August 2024 4,733 2 View
I am currently working on LncRNA; to know the lncRNA-protein interactions I want to do RNA pull down assay, so I need to design primers with T7 promoter. I need assistance in this regard.
07 August 2024 6,622 1 View
I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...
07 August 2024 7,535 4 View