Reverse transcription synthesis of single-stranded cDNA experiment
Reverse transcription synthesis of single-stranded cDNA experiment
The volume of a cDNA synthesis reaction is dependent on the number of anchored-anywhere primer combinations used. The protocol provided below, applies to 24 primer combinations and two samples. For more samples and/or more primer combinations, adjust the volume of the corresponding body mix. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Lijia Qu.
Operation method
Reverse transcription synthesis of single-stranded cDNA experiment
Materials and Instruments
RNA RT Body Mix Move I. Materials For more product details, please visit Aladdin Scientific website.
Thin Wall PCR Tubes Centrifuge Tubes Thermocyclers
1. Nucleic acids and oligonucleotides
RNA from Scheme 2
For each individual H-T11M primer, separate RT body mixes were prepared
Distilled water 28.2ul
5X RT buffer 12.0ul
2.5mmol/LdNTP mix 4.8ul
H-T11M (originally H-T1M. One translator changed)
Primer (here M=G, A or C) 6.0ul
2.Specialized equipment
0.5ml centrifuge tube
0.2ml thin-walled PCR tube (GenHunterT101)
Thermal cycler [GeneAmpPCRSystem9600 (Perkin-ElmerCorporation, Norwalk, CT) or Mastercycler (EppendorfScientific, Westbury, NY) recommended]
3. Additional reagents
RNAspectraFluorescentmRNA Differential Display System (GenHunterF501-F510R501-R510), including distilled water, 5XRT buffer [125 mmol/L Tris-HCl, pH 8.3, 188 mmol/L KCl,7.5 mmol/L MgCl2. 25 mmol/L dithiothreitol (DTT)], FDDdNTP mix (2.5 mmol/L), anchoring primer (H-T11M) (2 umol/U, and MMLV reverse transcriptase (100 U/ul)
II Methods
cDNA synthesis
1. Prepare 3 reaction tubes (one for each of the 3 H-T11M primers) according to the steps below.
Take 42.5ul of RT body mix for each and add to a labeled, RNAase-free 0.2ml thin-walled PCR tube. Remember to prepare separate RT reactions for each anchoring primer ( H-T11M ) used!
2. Using DEPC-treated H20, dilute the RNA to a final concentration and mix well, place on ice.
3. Take 5.0ul of diluted RNA (0.1ug/ul),add to a 0.5ml tube and mix well.
4. Set up the thermal cycler according to the following program: 65°C 5min→37°C 60min→75°C 5min →4°C equilibrium.
5. Place the reaction tube in the thermal cycler and start the program.
6. When the reaction reaches 37°C for 10min, pause the thermocycler, add 2.5ul of MMLV Reverse Transcriptase to each tube, mix quickly and continue to incubate.
7. At the end of reverse transcription, centrifuge briefly at maximum speed and collect condensate.
8. Place the reaction tubes on ice or at -20°C and set aside.