PCR amplification of double-labeled signatures
PCR amplification of double-labeled signatures
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
PCR amplification experiments with double-labeled signatures
Materials and Instruments
Solutions and Buffers LoTE cDNA Labeling Move Experimental Programs A and B I. PCR amplification of ligand mixtures 1. Add LoTE to increase the volume of the ligation mixture to 20ul. 2. Dilute the linkage mixture at 1% concentration. 3. Use Iul of 1% diluted linkage mixture as template in a 50ulPCR reaction: 4. Finally, add 1ul of diluted linkage mixture to the PCR reaction as template. 5. Use the following conditions for PCR amplification: After PCR amplification, analyze IOul of PCR mixture by electrophoresis on a 12% polyacrylamide gel, using an IOul ladder as a marker, which we recommend using the Mini-PROTEANII Vertical Electrophoresis System for electrophoresis (BioRad). 1. After bulk amplification of the diluted ligation mixture (100XIOOul of PCR reaction), mix in a 100 ml beaker (mixing volume of about 10 ml). 2. 50 ml of PB buffer (QIAquickPCR Purification Kit) in diluted PCR mixture, mix well. 3. Add 600ul to each of the 16 QIAquick columns. 4. Centrifuge in a microcentrifuge at 13000r/min for 45s. 5. Pipette the filtrate and repeat 5 times with the same column, each time adding 600ul of diluted PCR mixture. 6. After the last filtrate is removed, centrifuge once to remove all liquid. 7. Rinse with 0.75 ml of buffer PE (QIAquickPCR Purification Kit). 8. Centrifuge at 13000r/min in a microcentrifuge for 45s. 9. Dispose of the flow-through liquid and spin the Imin again at full speed to remove all liquid. 10. Place the column into a clean 1.5 ml Eppendorf tube. 11. Add 30ul of elution buffer (QIAquickPCR Purification Kit) to one column. 12. Leave at room temperature for 15 min. 13. Centrifuge Imin at 13OOOr/min in a microcentrifuge to elute DNA. 14. Mix the DNA eluted from the 16 columns into one tube (total volume 480ul). For more product details, please visit Aladdin Scientific website.
