FDD-PCR experiment

Summary

This protocol is designed as 24 PCR reactions using 3 cDNA subpopulations (RT reactions utilizing the H-T11M primer from protocol 3) with primers that are a combination of a fluorescent dye-labeled anchoring primer (FH-T11M) and 24 upstream arbitrary primers (HAP). This experiment was derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.

Operation method

FDD-PCR experiment

Materials and Instruments

cDNA Mineral Oil
Thermal cycler Thin-walled PCR tubes

Move

I. Materials

1. Buffers, solutions and reagents

Mineral oil (optional)

2. Nucleic acids and oligonucleotides

cDNA from Scheme 3

3. Specialized equipment

Thermal cycler [GeneAmpPCRSystem9600 (Perkin-Elmer) or Mastercyder (EppendorfScientific) recommended].

0.2 ml thin-walled PCR tubes

4. Additional reagents

RNAspectraFluorescentmRNA Difference Display System (GenHunterF501~F510&R501-R510), including distilled water, 10XPCR buffer (100 mmol/LTris-HCl, pH 8.4, 500 mmol/LKC1, 15 mmol/ _LMgCl2,0.01% gelatin), FDDdNTP mix (2.5 mmol/L), fluorescent anchored primer (FH-TUM) (2umol/L), and arbitrary primer (H-AP) (2umol/L)

II. Methods

1. In separate 1.5 ml centrifuge tubes, a separate FDD-PCR Master Mix was prepared for each FH-TUM anchoring primer.

Distilled water 91.8ul

10XPCR Buffer 18.0ul

FDDdNTP mix 14.4ul

FH-T11M Primer (M=G, A or C) 18.0ul

Taq DNA polymerase (Qiagen201207Valencia, CA) 1.8ul

2. Program the thermal cycler to 94°C15s→40°C2 min→72°C60s→40 cycles→4°C equilibrate.
If a non-recommended thermal cycler is used, adjust the denaturation (94°C) 'time to 30s.

3. Take 16ul of each of the corresponding FDD-PCR Master Mix and add to labeled 0.2 ml thin-walled PCR tubes. For example, label 8 tubes as a combination of the anchor primer _FH-T11 G with any primer H-AP1~8, and similarly label all 24 primer combinations, including FH-T11A with H-AP1~8 and FH-T11C with H-AP1~8.

4. In each 0.2 ml PCR tube, add 2.0ul of the corresponding cDNA (from the RT reaction of Protocol 3).

5. To each 0.2 ml PCR tube, add 2.0ul of the corresponding H-AP, mix well, and make a total reaction volume of 20ul. 25ul of mineral oil will be added if required.

6. Place the 0.2 mlPCR tubes in the thermal cycler and start the program (see step 2). When finished, store the reaction products in the dark at -20°C.

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