I am using transwell plates for my chemotaxis assays. Now I am planning to switch over too Boyden chamber but no one in my lab seems to have used it before. Does anyone know where can I find any training for using these Boyden chambers.
Thank you very much for your reply. how would I pick one cell from an aliquot of cells and how would I grow them? Is there any protocol for that? I thawed three frozen vials of a cell lines and grew them separately. Would you consider them as three clones?
I am looking at BCR/ABL expression of two philadelphia positive cell lines. According to plan, I have to use three clones of each cell line and do a PCR in triplicates. How long do I have to grow a cell line in three different flasks to call them different clones of a cell line? Is there any guideline or definition
1. I have to do a Taqman PCR on these samples, but will need to do SNP on some special samples after the PCR. Is a different type of DNA needed for SNP array?
2. I have cerebrospinal fluid samples banked in Trizol. I have about 600 samples already collected. For new samples, I have already started using Qiagen DNA Micro kit which is optimized for small quantity of samples.
3. I want to optimize the method for extraction of the samples already stored in TRIZOL. I have a very low yield of DNA and a very low 260-280 and 260-230 ratios.
A happy Christmas and new year!
I am using TRIZOL to extract DNA from human cerebrospinal fuid (CSF). Generally, the number of cells in the CSF are very low (100-1000 cells/ml) and I am having trouble with the yield and quality of DNA. I use 800ul Trizol, 1 ul glycogen just before ethanol precipitation, and Na-acetate to wash the pallet. Then I use 75% ethanol to remove the salt and then resuspend in water. In the original protocol, resuspension should be with 8mM NaOH. Is this necessary or can I use water to resuspend?
Today, I made a mistake and instead of using 75% EtOH, I used 25% EtOH to wash the pellet. Since, the quantity of DNA is so low that I usually cannot see a pellet, and neither I can use a spectrophotometer to quantify the DNA, I am not sure if I have lost the DNA.
Besides this, does someone have a general advice to increase the yield of DNA from extremely low number of cells? These are human samples and I cannot play around.
P.S. In Na-acetate wash step, can I just do it once instead of two washes? I fear with each wash step I will loose DNA. Also, would 10% ethanol in Na-acetate solution not dissolve the DNA?