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.
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
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
@John Hildyard i tried many times but RNA is getting degraded after DNase treatment, i am using Thermoscientific DNase 1
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
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