It is a complementary experiment to experimental protocols A and B of the differential display technique (DD) experiment and the gene expression series analysis experiment.
Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Z. Q. Zhao Jun Chen
Operation method
Separation experiments with double labels
Materials and Instruments
Solution & Buffer BSA LoTE cDNA Labeling Move I. Digestion of the double label with NlaIII to release the joints 1. Add the following to 480ul of purified PCR product: 2. Digest at 37°C for Ih (do not heat inactivate the enzyme as this will denature the 22-26 bp small doublet). 3. Digested products can be stored overnight at 4°C or biotinylated PCR products can be extracted using a Dynabead. Note: In this step, the reaction product is enriched due to the capture of biotin streptavidin anti-biotin, which removes the junctions and unenzymatized or partially enzymatized products. 1. Swish the Streptavidin anti-biotin M-280 Dynabead for 1 min to bring it back into suspension. 2. Transfer 3 IOOul of Dynabead into a 1.5 ml Eppendorf tube. 3. Fix the beads with a magnetic device and discard the supernatant. 4. Resuspend and rinse 3 times with 200ul of IX binding and rinsing buffer, immobilize the beads and discard the rinse solution. 5. Enzymatically dissolve NlaIII double label (volume 600ul) into three 200ul portions; add 200ul to each of the three rinsed beads. 6. Mix well and incubate at room temperature for 30 min (mixing every IOmin). 7. Fix the beads with a magnetic device and transfer the supernatant into a new 1.5 ml Eppendorf tube. 8. To precipitate the supernatant, it is necessary to add: 1 3ul of glycogen 800ul Ethanol 9. Ice bath on dry ice/ethanol for 15 min. 10. Centrifuge in a microcentrifuge at 13000r/rnin for 15 min at 4°C. 11. Wash the precipitate with ethanol once and air dry. 12. Freshly suspend it in 30ul of LoTE. 13. Sample the entire sample on an 8 lane I2% polyacrylamide gel, using the IObpladder as a molecular weight standard (note: we recommend using a sampling buffer containing Orange G, which prevents co-movement with the 22-26bp double label). 14. Perform gel electrophoresis for 2.5 h at room temperature and 50 V, or until yellow G is at the bottom of the gel. 15. EB staining. 16. A double labeled band of 22 to 26 bp is excised from the coagulation strand (Fig. 2-5), which should be below the junction at 40 bp. 17. Dispense the cut polyacrylamide gel fragments into four 0.5 ml PCR tubes, each of which is perforated at the bottom with a 21-gauge needle. The PCR tubes were placed into a 1.5 ml Eppendorf tube and centrifuged in a microcentrifuge at 13,000 min for 5 min to fragment the polyacrylamide gel. The 0.5 ml PCR tubes were removed, and 300ul of LoTE was added to each tube, vortexed, and incubated at 37°C for 15~30 min. 18. Transfer the polyacrylamide gel suspension to a SpinX column and spin in a microcentrifuge at 13000r/min for 5 min. 19. Pipette each filtrate into a new 1.5 ml Eppendorf tube and extract with an equal volume of PCI (protocol A, step 2). 20. Add the following to precipitate the liquid: A lOOullOmol/L of ammonium acetate 3ul of glycogen 900ul of ethanol 21. Ice bath on dry ice/ethanol for 15 min. 22. Centrifuge in a microcentrifuge at 13000r/min for 5 min at 4°C. 23. Rinse the precipitate twice with 70% ethanol, then air dry. 24. Redissolve the 4 precipitates in a total volume of 7ul of LoTE. For more product details, please visit Aladdin Scientific website.
