| 1. How many samples can be analyzed per kit? Single reaction: Duplicate: 2. How much protein is needed for each assay? The working range for 293T cell extract or equivalent is 0.5 ng - 1000 ng per PCR reaction. For other proteins, use no more than 1µg per reaction. 3. How many dilutions are needed for each sample? We recommend that you do at least 3 serial dilutions for each sample. 4. Do you recommend double-testing the standard curve? It is not necessary to double-test the standard curve due to its sufficient stability; however, if desired it can be done at the beginning of the experiment until the user is comfortable with the assay. 5. For how long do you incubate the cells with the lysis buffer and at what temperature? For 6-well plate: · Wash cell with 2 ml PBS per well, RT. Add 0.5 ml Trypsin to each well, incubate 5min at 37C. · Add 3 ml cell culture medium to each well and harvest to 15 ml tube. · Centrifuge at 1000 x rpm, 5min at 4C. Remove supernatant and add 3 ml cold PBS washing. · Centrifuge at 1000 x rpm, 5min, 4C. Remove PBS. · Add 200ml of 1 X Lysis Buffer per 105-106 cells, mix well and incubate for 30min at 4C. · Centrifuge, 13,000 x rpm, 30min at 4C. · Transfer the supernatant for analysis. 6. Is the 1 X Lysis Buffer compatible with BCA protein assay reagent? Yes. You also can use RC DC kit from Bio Rad to measure the protein concentration. There are no components in the buffer that will interfere with the measurement. 7. What can we do if the provided 1 X Lysis Buffer is not enough? We sell the 1 X Lysis Buffer separately. Catalog # MT3002. Or you can stay with the lysis buffer you are currently using to prepare your samples, and then use the provided buffer to make the dilutions. 8. Which fluorescent should be selected on our real-time PCR machine? Select FAM-490 on the PCR machines from most vendors, such as Bio-Rad, Stratagene. For others, please consult manufacture for specifications. 9. What is the purpose of the "melting curve" step and when should it be performed? The melting curve step is to verify the content of PCR product. The melting point is the denature temperature of double-stranded DNA. The longer the DNA fragments, the higher the melting temperature. Primers may form dimmers and their melting points are different from the amplified DNA fragments. 10. How stable is the QTD kit? It is quite stable (up to several days at room temperature). 11. How long is the shelf life of the QTD kit? For at least 1 year at -20C. 12. Can PCR products be analyzed by PAGE? No, not at this point. 13. I expect that my cells express very low levels of telomerase activity. Can I try to make a more "concentrated" sample by using less lysis buffer? Yes. 14. Can the result be quantified by comparing with TSR standard? TSR standard Ct value gives a reference for quantification. 15. I was not able to reach the same standard curve of TS R as appears in the protocol and also the results were not reproducible. In every test I got a different CT. Try to adjust the threshold baseline to be the same for every test. |