I use ABI one step qRT-PCR system. I am hesitating between two ways of setting up the final reaction mixture.
1) prepare a tube of mixture for triplicate reaction for each sample. The final step is to add 20ul final reaction mixture into the 384-well plate;
2) prepare a master mixture for all reactions; add RNA samples (in triplicate) into the 384-well plate. The final step is to add the aliquot of master mixture into the wells which already have RNA samples in them.
I felt the second way is easier than the first way, especially when I have many RNA samples and quite a few genes of interest. But with RNA samples in the 384-well plate (= open to the air) all the time until addition of the master mixture, I worry about any possible contamination/degradation.
Which way do you guys use? I'd appreciate any suggestions on this issue! Thanks in advance!
I need to prepare cell suspensions of seven cell lines and use them at the same time for some experiment. Preparation for each cell line takes at least 30min. So I am wondering where to keep the prepared cell suspensions when working on others. Should I keep them on ice, on bench at room temperature, or 37C/5% CO2 incubator? Can they be stable for several hours?
I need to do check proliferation of my cell lines in serum free condition. So I have two choices:
1) trypsinize cells and resuspend cells in 10%FBS medium, plate cells; change medium to serum free the next day;
2) after trypsinizing and resuspending in 10%FBS medium, centrifuge cells, aspirate 10%FBS medium; resuspend cells in serum free medium and plate cells at desired amount.
I would prefer choice 2) since washing and changing medium may cause loss of this specific cell line. But I don't know if cells can adhere to the plate when plated in serum free medium.
When I read papers, I noticed that sometimes authors mentioned about the cell confluency. For example, they may isolated RNA for microarray experiments "when cells are about 60% confluency"; they may collect protein lysates (to check phosphorylation level) "when cells are about 80% confluency". Why cell confluency matters? Sometimes to have as much lysates as possible, I let cells grow to almost complete confluency, would that affect my results?
I have been trying to do soft agar assay, which is a pain to me, especially when I have several cell lines. I have a couple of questions. I hope experts here can help me out.
Question 1: I saw people use agarose on some protocols. Is that the same thing as agar?
Question 2: how many times I may reheat the agar without affecting the experimental result? When I have several cell lines, the remaining top agar always set after I was done with the first cell line. I am wondering if I may reheat it again and again.
Question 3: after adding the top agar with cells onto the bottom agar, should I put the plates at 4C to solidify or just 37C incubator?
Question 4: what do you guys use to visualize the colonies? I have seen crystal violet or MTT on different protocols. Any preference? what concentrations for each? Time of incubation?
I am using RNeasy kits from Qiagen for RNA isolation. I am wondering if the samples could be frozen down after lysis step. If yes, stored at what temperature? What risks are associated for long-term storage?