Question

please , i need Write the protocol by your own words ( steeps)

5. Add 1 volume of 70% ethanol to the cleared lysate, and mix immediately by pipetting. Do not centrifuge. Proceed immediately to step 6. Note: The volume of lysate may be less than 350 pl or 600 pl due to loss during homogenization and centrifugation in steps 3 and 4. Note: Precipitates may be visible after addition of ethanol. This does not affect the procedure. 6. Transfer up to 700 pl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at 28000 xg (210,000 rpm). Discard the flow through. Reuse the collection tube in step 7. If the sample volume exceeds 700 pl, centrifuge successive aliquots in the same RNeasy spin column. Discard the flow-through after each centrifugation." Optional: If performing optional on-column DNase digestion (see "Eliminating genomic DNA contamination", page 26), follow Appendix D (page 82), steps 1-4, after performing this step. 7. Add 700 pl Buffer RW1 to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at 28000 x g (210,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 8. Note: After centrifugation, carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through. Be sure to emply the collection tube completely Skip this step if performing optional on-column DNase digestion (page 69).

Answer 1

5. Take the lysate from centrifuge that has the volume of 350 ul to 600 ul add 1 volume that is 3.5 ul or 6 ul of 70 percentage ethanol to the lysate solution and mix it well by pipetting using pipete

6. To remove the other contamination add 700 ul of lysate mixed up with 70 percentage alcohol precipate into RNease column that is kept in 2 ml collecting tube and centrifuge at 8000 rpm for 15 seconds. Discard the flowthrough collected in the collecting tube . Repeat the same if extra sample is present

7. Add 500 ul of RPE buffer in RNease spin column and centrifuge at 800 rpm for 15 seconds and discard the collecting tube flow through

8. Add 500 ul of RPE buffer again to the spin column and centrifuge as mentioned in step 7 and discard the flow through

9. Add 500 ul of RPE buffer again to the spin column, do centrifuge as mentioned in step 7 and discard the flow through without disturbing the column

10. Fix a new 2 ml collection tube in the Rnsea soon column and centrifuge to remove excess RPE buffer

11. Place the RNease spin column in new 1.5 ml collecting tube and add 30-50 ul of RNase free water close it and do centrifuge for 1 minute at 8000 rpm to elute the RNA

12.repeat the step 11 to get RNA yield more than 30 ug.