Designing and Preparing dsRNA: Difference between revisions
mNo edit summary |
mNo edit summary |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 9: | Line 9: | ||
==Preparing dsRNA== | ==Preparing dsRNA== | ||
===Template Preparation=== | ===Template Preparation=== | ||
*set up a 250 uL PCR reaction | *set up a 250 uL PCR reaction | ||
| Line 19: | Line 17: | ||
**Template (previously prepared PCR product or 1 uL of cDNA) | **Template (previously prepared PCR product or 1 uL of cDNA) | ||
**2 uL KOD polymerase | **2 uL KOD polymerase | ||
**Water | **132uL Water (bring up to 250 uL) | ||
**Run using TD-KOD program | **Run using TD-KOD program | ||
*transfer PCR product to a clean tube. Add 25 uL of 3M NaAcetate (pH 5.2) and 500 uL ice cold ethanol (stored at -20). | *transfer PCR product to a clean tube. Add 25 uL of 3M NaAcetate (pH 5.2) and 500 uL ice cold ethanol (stored at -20). | ||
| Line 40: | Line 38: | ||
*Dilute RNA 10x in loading dye | *Dilute RNA 10x in loading dye | ||
*Load 1 and 3 uL of diluted RNA, plus 4 or 8 uL of 1 kB Plus DNA Ladder | *Load 1 and 3 uL of diluted RNA, plus 4 or 8 uL of 1 kB Plus DNA Ladder | ||
*Using ImageJ, quantify ladder and dsRNA bands and calculate concentration. Check that the dilutions are close to 3x apart in signal intensity. | *Using ImageJ, quantify ladder and dsRNA bands and calculate concentration. See [[Using ImageJ to Quantify Bands]]. Check that the dilutions are close to 3x apart in signal intensity. | ||
*Label RNA with concentration and store at -20 | *Label RNA with concentration and store at -20 | ||
*see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html | |||