CDNA Synthesis Protocol

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Current revision as of 19:34, 6 March 2009

Contents 1 Things to Remember 2 Calculate RNA needed for Each RT Reaction 3 Reverse-Transcription (RT) Reaction 4 Determine Concentration of cDNA 5 cDNA Synthesis Information Table

Things to Remember

1. Be sure we have the required reagents before you start (see below)
2. Always use new gloves and change them frequently
3. Get ice!
4. Thaw Oligo dT, RNase-free dNTPs, and 5x 1st Strand buffer at RT on kimwipes and DTT on ice RIGHT NOW!!!...allow at least ~20min to thaw completely
5. Water bath to 42C (RT reaction)
6. Dry bath to 65C (RT reaction)
7. If not performed directly after RNA Extraction Protocol, then sterilize the working area with RNase Zap wipes (wipe once with wipe and then use RO water from bottles to rinse with brown paper towels, and repeat another RO water rinse and towel wipe)…if anything gets touched with non-RNase sterilized gloves/hands/sleeves/etc., then this process must be repeated for those contaminated surfaces throughout the protocol
   1. PCR bench area
   2. tabletop minicentrifuges, microcentrifuges, dry baths
   3. pipetmen & pipet tip barrier tip boxes
   4. plastic eppendorf tube holders
   5. pens (sharpies and pens for notes)
8. RNase-free prelabeled tubes (~3-4/reaction) and RNase-free H2O (check that enough in bottle in the open RNeasy Plant Mini kit)

Calculate RNA needed for Each RT Reaction

• Want to use ~1ug (1000ng) of RNA per RT rxn, 10uL is the max volume

Reagent	~ value ng/uL of sample	900-1000	500	330	250	200	165	140	125	110	1-109
Oligo dT (500ug/mL)	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL
1ug of RNA (up to 10uL)	xuL	1uL	2uL	3uL	4uL	5uL	6uL	7uL	8uL	9uL	10uL
dNTP mix	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL
RNase-free water (up to 9uL)	xuL	9uL	8uL	7uL	6uL	5uL	4uL	3uL	2uL	1uL	0uL
Total Volume	12uL

Reverse-Transcription (RT) Reaction

• Check waterbath and dry bath that they are at the correct temperatures
• Do not start until temperatures are correct
• Be sure Oligo dT, RNase-free dNTPs, and 5x 1st Strand buffer at RT on kimwipes and DTT on ice are completely thawed…flick to mix and do a quick spin down of each just prior to use

RT Reagent Values for Your RNA Samples

3code
Oligo dT (500ug/mL)	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL
1ug of RNA (up to 10uL)	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL
dNTP mix	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL	1uL
RNase-free water (up to 9uL)	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL	___uL
Total volume	12uL	12uL	12uL	12uL	12uL	12uL	12uL	12uL	12uL	12uL	12uL	12uL

RT Step 3 Master Mix

Reagent/# Reactions	1x (uL)	____x (uL)
5x 1st-strand buffer	4uL
0.1 M DTT	2uL
RNase-free water	1uL
(add 7uL of MM to each tube, changing tip each time)

1. In prelabeled 1.5mL RNase-free tube, combine the reagents from the RT Reagent Values for Your RNA Samples Table…flick to mix and do a quick spin down of each just prior to use… above for each individual RNA reaction to undergo RT into cDNA in their respective tubes and briefly centrifuge using tabletop centrifuge
   1. 3code, cDNA1 or cDNA2 (depending on which RNA rinse was used to make it), date, initials, on top and side!
   2. Only do as many as you can quickly process/pipet at a time…may need to split into staggered sets of 4-6 reactions at a time
2. Heat tubes at 65C in drybath for 5min and then quick chill on ice (at least 1min)
   1. PUT RNA AT -80C NOW
   2. Make RT Step 3 Master Mix as per your table calculations above (allow your expected # of reactions + 1-2 extra reactions to account for any pipetting errors) in a prelabeled 1.5mL RNase-free tube
   3. Briefly centrifuge using tabletop centrifuge
   4. Can remain on ice for up to 30min
   5. Increase drybath to 70C for last step (heat inactivation)
3. After MM is made, flick to mix and do a quick spin down and add 7uL of MM (change tip each time) to each cDNA tube after out of 65C and chilled
4. Gently flick to mix and incubate for 2min at 42C in water bath
5. Add 1uL of SuperScript II RT (in blue labtop cooler in -20C freezer) and mix by gently pipetting up and down
6. Incubate at 42C for 50min in water bath…centrifuge briefly after ~25min and again after full time to bring any condensation back into the reaction liquid
7. Heat inactivate the reaction at 70C for 15min in the dry bath

Determine Concentration of cDNA

• Follow Nanodrop Protocol
• Nucleic acid type should be left at DNA
• Use RNase-free water next to ND-1000 as blank
• Load 1uL of each sample onto Nanodrop (very precious, don’t waste unnecessarily!)
• Take note of curves
• Use one of your pre-labeled 1.5mL test tubes for a dilution after you have estimated your cDNA value (generally 1:10 dilution; 45uL RNase-free water into RNase-free tube + 5uL cDNA that you just nanodropped)
  • Tube Label = 3code, cDNA1 or cDNA2 (depending on which RNA rinse was used to make it), date, initials, dilution ratio; on top and side!
• Store cDNA at -20C

cDNA Synthesis Information Table

ORGANISM NAME	3CODE	RNA TUBE INFO (Person, Date, Rinse#, etc.)	RNA COMMENTS (ND quality, etc.)	cDNA NANODROP COMMENTS (curve, amount, quality)	OTHER NOTES