Hi,
I extract RNA from different plant tissues but RNA concentration when check on nanodrop varies too much from each other e.g one sample concentration is 3066 ng/ul and other is 6000 ng/ul but i want to dilute it to have all samples RNA concentration to same point e.g 500 ng/ul, so in this way cDNA have same concentration. So I want to know if there is any formula i can use to make the same RNA concentration? I did manually but i did not work. Please need help
I'm not sure what you mean it did not work? IF you use the basic dilution equation concentration1 x volume1=concentration2 x volume2 then solve for volume2 and add RNAse free water to volume 2 but be sure to account for the starting volume. This will get you approximately equal concentrations but if your samples are not very pure you can still have some variability.
@Sarah Zilka according to this equation volume 1 is how much RNAse water i add, concentration 1 means my RNA 1st concentration after extraction. I am confuse about concentration 2 and volume 2, what it mean, can you please explain , thanks
volume1 is the starting volume of RNA. Concentration1 is the concentration of RNA from the nanodrop. Concentration2 is the desired concentration in your case 500 ng/uL. solve for volume2.
So using one of the concentrations you used if you have 20 uL of the 6000 ng/uL RNA
(20uL x 6000 ng/uL)/500ng/uL=240 uL
240 uL is the final volume you want so then to determine the volume of water you should add subtract the starting volume of RNA in my example 20 uL
240 uL-20 uL=220 uL
So you would add 220 uL water to the sample to obtain a 500 ng/uL solution.
Seems like you got very good RNA i always gets RNA of low quality like 300-400 ng/ul.. Would you please share your protocol on [email protected].
Thanks !
@Harshraj Subhash Shinde
HOMOGENIZATION:
1. Prepare 1.5ml Safe-lock eppendorf tubes and add 5-6 glass-beads.
2. Add 4-6 young leave or (any tissue) to each tube and freeze immediately in liquid nitrogen.
Samples can be used immediately or stored for up to several months at -80°C.
3. Cool these tubes in liquid nitrogen for 5-15 min.
4. Homogenize the tissue in a Geno grinder or mortar and pestle for at least 5 minutes to make it completely crushed.
RNA extraction:
5. Keep out tubes one by one from liquid nitrogen and immediately add 1ml (1000 ul) of TRIZOL to the homogenized tissue and mix it very well for 1 min by vortexing.
6. Incubate at Room Temperature for 5 min.
7. Centrifuge the samples at 12000 rpm for 10min at 4°C.
8. Transfer the supernatant to new RNase free tubes.
9. Add 0.2ml (200 ul) of trichloro methane (chloroform).
10. Shake vigorously by hand or vortex it for 30 seconds.
11. Incubate at RT for 3 min.
12. Centrifuge the samples at 12000 rpm for 10min at 4°C for phase separation.
13. Transfer the aqueous upper phase to new tubes from other three phases in tube.
14. Precipitate RNA by mixing with 0.5 ml (500 ul) isopropanol per 1ml TRIZOL and gently mix by hands.
15. Incubate at RT for 30 minutes OR keep it at -20°C for two hours OR keep it O/N at -20°C.
16. Centrifuge the samples at 13000 rpm for 10min at 4°C to obtain pellets
RNA wash:
17. Remove supernatant and wash pellets with 1ml 75% EtOH (diluted with
DEPC treated water).
18. Vortex once and centrifuge at 7500 x g for 5min at 4°C
19. Discard supernatant and dry pellets for 5 min at RT (or in speed-vac)
20. Dissolve pellets in DEPC treated water
21. Incubate at 55°C for 10 minutes.
Store at –80°C until use.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA