Hi everybody
I want to extract RNA from human whole blood. but I have a problem. my 260/280 absorption is low(about1.2).while I had good results for another sample(such ticks, about 1.9).so I think my problem is with RBC lysis buffer.am I right?
if so...
1)how can I produce good RBC lysis buffer for a high quality of RNA?
2)can I use off ficoll for lysate?
Genomic Medicine Biorepository
GMB003
Protocol GMB003rB: 20090216NP
Revised and approved 2012JULY26 by B. Sebastian
24) Allow the pellet to dry for 5 to 10 minutes to remove any remaining ethanol.
25) Dissolve the RNA pellet by adding 20 µl of RNAse-free H2O to each sample.
26) RNA should be quantitated within 2 hours of elution. It can be kept at 4 C until that time;
it can also be held temporarily at -20 until permanent storage at -80. Repeated freezethaws are to be avoided, so RNA should be aliquoted for transfer as soon as possible after
quantitation.
10x RBC Lysis Buffer
89.9 g NH4Cl
10.0 g KHCO3
2.0 ml 0.5 M EDTA
Dissolve the above in approximately 800 ml ddH2O and adjust pH to 7.3. QS to 1 liter and mix
thoroughly. This solution is stable for 6 months at 2 – 8° C in a tightly closed bottle.
1x RBC Lysis Buffer
Simply dilute the 10x stock solution 1:10 with ddH2O. Stable for 1 week at room temperature.
TRIzol Reagent
Invitrogen Life Technologies: Cat No. 15596018
OR
RNA STAT-60 Reagent
Tel-Test: Cat No. CS-111
Other Reagents Needed
Phosphate Buffered Saline (PBS)
Isopropanol (2-propanol)
Ethanol
RNAse-free water
RNAse-Away (a cleaning solution that neutralizes RNAses on bench tops, pipettors, centrifuges,
and other equipment.
You could also consider doing DNase treatment if applicable to your protocol, i always find it helpful in improving 260/280 absorption .
Thank you so much for your suggestion.
If there is another offer, I would appreciate it.
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