Re-amplification, cloning and sequencing experiments of differentially expressed cDNAs
Re-amplification, cloning and sequencing experiments of differentially expressed cDNAs
After cutting the latent differentially expressed cDNA from the acrylamide gel, the cDNA is re-amplified with the same anchored-any primer combination under the same reaction conditions as the initial PCR. The re-amplified product can be cloned and sequenced for further analysis. This experiment is based on the PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Re-amplification, cloning and sequencing experiments of differentially expressed cDNAs
Materials and Instruments
Agarose gel Distilled water DNA spotting dye Glycerol Distilled water Bromophenol blue Xylene cyanide FF Ethidium bromide solution Arbitrary primer (H-AP) cDNA dNTP mix Unlabeled anchored primer Carrier-specific primer PCR Buffer Tris-HCl KCl MgCl2 Gelatin TaqDNA polymerase Move I. Materials For more product details, please visit Aladdin Scientific website.
Electrophoresis unit Centrifuge tubes Thermal cycler Thin-walled PCR tubes UV transilluminator Microwave oven
1. Buffers, solutions and reagents
1.5% agarose gel with ethidium bromide (see step 9)
distilled water
DNA spotting dye
30% glycerol
70% distilled water
0.003% Bromophenol Blue
0.003% Xylene cyanide FF (GenHunterS403)
Ethidium bromide solution (0.5ug/ml)
Distilled water
Re-amplification mix (see step 7)
2. Nucleic acids and oligonucleotides
Arbitrary primer (H-AP) (2 mmol/L)
cDNA (cut strips) from Scheme 5
dNTP mix (250 umol/L) (GenHunterS501)
Unlabeled anchoring primer (H-T11M) (2umol/L)
Carrier-specific primers [Lseq/Rseq(GenHunter)
3. Enzyme and enzyme buffer
10X PCR buffer
100 mmol/L Tris-HCl, pH 8.4
500 mmol/LKCl
15 mmol/LMgCl2
0.01% gelatin
TaqDNA polymerase (Qiagen201207)
4. Specialized equipment
Electrophoresis device
1.5 ml centrifuge tube
Parafilm
Thermal cycler
0.2 ml thin-walled PCR tubes (GenHunterT101)
UV Transilluminator
Microwave oven
5. Additional reagents
PCR-TRAPCloningSystem (GenHunterP404), including PCR-TRAP cloning vector ready for insertion, T4DNA ligase (200 u/ul), distilled water, 10X ligation buffer (500 mmol/LTris-HCl, pH 7.8, 100 mmol/LMgCl2. 100nimol/LDTT, 10 mmol/LATP, 500ul/ml BSA) and the GH-competent RNA spectra Fluorescent mRNA Differential Display System (GenHunterF501-F510R501-R510, or S201).
6. Cells
GH-competent cells
7. Special Projects
Agar plates containing antibiotics (refer to cloning system recommendations for specialized antibiotics and protocols)
II. METHODS
1. Place the cut strip from protocol 5 into a 1.5 ml centrifuge tube and add 1 ml of distilled water.
2. Soak for 30 min and vortex frequently.
3. Remove the distilled water (do not lose the strip) and add 50 ul of fresh distilled water.
4. Cap the tube tightly (seal with Parafilm) and boil for 15 min to wash the cDNA out of the gel.
5. Centrifuge for 1 min, concentrate and collect the gel.
6. Transfer the supernatant to a new 1.5 ml centrifuge tube and discard the tube containing the gel.
7. In a 0.2 ml thin-walled PCR tube, add the following components to complete the re-amplification of the cDNA.
Distilled water 22.6ul
10XPCR Buffer 4.0ul
dNTP mix (250umol/L) 1.0ul
H-AP Primer (2umol/L) 4.0ul
H-T11M (2umol/L) 4.0ul
cDNA template 4.0ul
Taq DNA polymerase 0.4ul
8. Place the re-amplified reaction solution on a thermal cycler and run the reaction under the original PCR conditions (see Scheme 4, step 1).
9. Prepare a 1.5% agarose gel containing ethidium bromide.
a. In a glass flask, weigh 1.5 mg of ultrapure electrophoresis-grade agarose.
b. Add 1XTAE to 100 ml.
c. Melt the agarose in a microwave oven.
d. After cooling, add 3ul of 1:1 ethidium bromide solution.
e. Mix well and pour into gel device.
f. Place the comb and wait for the gel to set.
g. The electrophoresis buffer is 1XTAE solution (the amount varies with the gel device).
10. In a 0.5 ml centrifuge tube, add 30ul of re-amplification reaction solution and 5ul of DNA spotting dye. Add the entire mixture to a 1.5% agarose gel.
11. Electrophoresis at 70V for about 45 min.
12. Observe with a UV transilluminator to confirm the cDNA re-amplification reaction product.
13. Clone the differentially expressed cDNA into the recommended PCR-TRAP cloning vector or other suitable vector according to the manufacturer's protocol.
14. If the PCR-TRAPCloningSystem is used, sequencing can be performed using the provided vector-specific primers. If other cloning vectors are used, refer to the manufacturer's sequencing instructions.