Alice:Single/2015-4-30

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SMARTer RNA seq protocol trial run #3[edit]

  • There are several possibilities where things might went wrong using Blue's old SMARTer kit to do previous trial run:
    • 1) the primers of Blue's old kit were hydrolyzed/reagents weren't fresh;
    • 2) the amount was too little to be detected/seen on gel (didn't run QPCR);
    • 3) the technique wasn't good enough to get the experiments to work, more practice needed;
  • In order to make sure the reagents were of good quality, Song purchased a new SMARTer v3 kit to do a trial run Link
  • Since this is a practice run, so we want to use less reactions if possible, I will use 1.5 kit reaction volume to run 3 reactions

Experimental Procedure[edit]

  • The Control RNA is supplied at a concentration of 1 μg/μl, it is diluted to 100 pg/ul for this practice run using serial dilution
  • reaction setup:
    • (A) 100 pg ctrl RNA
    • (B) 100 pg ctrl RNA + CDS primer IIA (IDT)
    • (C) 100 pg ctrl RNA repeat

First strand cDNA Synthesis[edit]

- Thaw all the reagents needed for first-strand cDNA synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store on ice.
- Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer - v3 with the RNase Inhibitor as indicated below (scale-up as needed), Vortex briefly to mix, then spin down:
volume component
19 μl 10X Lysis Buffer v3
1 μl Rnase Inhibitor
20 μl Total Volume
- Prepare each sample (10 μl total volume) in individual 0.2 ml RNase-free PCR tubes or in an 8-well strip.
Components Sample A Sample B Sample C
10X Reaction Buffer 0.5 ul 0.5 ul 0.5 ul
Nuclease-free water 3.5 ul 3.5 ul 3.5 ul
Diluted Control RNA 1 ul 1 ul 1 ul
Total volume 5 ul 5 ul 5 ul
- Place the samples on ice, and add 3’ SMART CDS Primer II A (12 μM) like below. Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube:
volume component
5 μl Cell/Total RNA in Reaction Buffer
0.5 μl 3’ SMART CDS Primer II A (12 μM)
5.5 μl Total Volume
- Place the tubes into a pre-heated thermal cycler and run 72°C for 3 min, then quickly put them on ice.
- Pre-heat the thermal cycler to 42°C again
- Meanwhile, prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature:
volume per reaction volume for 3 reactions component
2 μl 6ul 5X First-Strand Buffer
0.25 μl 0.75 ul DTT (100 mM)
0.5 μl 1.5 ul dNTP Mix (20 mM)
1 μl 3 ul SMARTer IIA Oligonucleotide (12 μM)
0.25 μl 0.75 ul RNase Inhibitor (40 U/μl)
1 μl 3ul SMARTScribe Reverse Transcriptase (100 U/μl)*
5 μl 15 ul Total Volume added per reaction
- Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it.
- Mix the Master Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube.
- Place the tubes in a thermal cycler pre-heated to 42°C. Run the following program:
- 42°C 90 min -> 70°C 10 min -> 15°C forever
- STOPPING POINT: The tubes can be stored at 4°C overnight.

cDNA amplification by LD PCR[edit]

- Thaw all the reagents needed for PCR (except the enzyme) on ice. Gently vortex each reagent tube to mix and spin down briefly. Store on ice.
- Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents in the order shown.
- NOTE: Remove the DNA polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly in a microcentrifuge to collect the contents at the bottom of the tube:
volume per reaction volume for 3 reactions component
12.5 ul 37.5 ul 2X SeqAmp PCR Buffer
0.5 ul 1.5 ul PCR Primer II A - v3 (12 μM)
0.5 ul 1.5 ul Nuclease-free water
1.5 ul 4.5 ul SeqAmp DNA Polymerase
15 ul 45 ul Total Volume added per reaction
- Mix well and briefly spin in a minicentrifuge to collect the contents at the bottom of the tube(s).
- Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program:
- 95°C 1 min -> ( 98°C for 10 sec -> 65°C for 30 sec -> 68°C for 3 min ) x 40 cycles -> 72°C 10 min

Discussion[edit]

  • The QPCR curve never came up. Then I realized that SYBR Green was never added to the reaction. I will run a gel to check the products.