e-mail+to+Kathy+about+this+experiment+1-9-12


 * from: |||| Kurt Whittemore kurtwhittemore@gmail.com ||
 * to: |||| Kathryn Sykes  ||
 * date: |||| Mon, Jan 9, 2012 at 4:42 PM ||
 * subject: |||| whole cDNA library ||
 * mailed-by: |||| gmail.com ||

Working with the whole cDNA library has been a learning experience. I don't have as many of the 3,000 spots that I wanted, but I do have 200 of the eventual 3,000 spots. Below I have some of the main issues and possible solutions to them.

Transformation Efficiency The transformation efficiency of the pooled 23 transformations was only 9.8*10^5 cfu/ug whereas in the past for one transformation the efficiency was 1.3*10^7 cfu/ug. The samples were only allowed to incubate for 40 min as they should. I still have quite a bit more DNA from the 1st ligation that I can use for more transformations, and then I can also use the B and C reactions later too. I think the decrease in efficiency was due to a delay in timing for one sample since there were so many samples. I'll see if this is true by just transforming one single sample to observe what kind of transformation efficiency I obtain. If it is high again, then I plan to transform 5 samples at a time in the future rather than 23. However, I was thinking I would transform, get the sample to the incubator right away, start a 40 min timer for that sample, transform the next sample, get the sample to the incubator right away, start a separate 40 min timer for that sample, and so forth. Then as the 5 different 40 min timers went off, I would put the samples in the fridge. When they were all done, I would pool them. Then I would use this pool to determine the transformation efficiency.

Dilution and Separation of Cultures When I worked with the cultures this time, I diluted them without knowing the transformation efficiency first. Therefore, I thought I was diluting and separating the culture so that there were 1,000 colonies per well. However, I later found out that I only had 79 colonies per well. I then pooled wells to go from 26 plates down to about 2 plates so that I had 1,000 colonies per well. This way I would later be able to fit as many colonies onto my nitrocellulose slide as possible. This was extremely inconvenient. I've seen that in many similar types of protocols they put the transformation solution in the fridge, and then they don't proceed with it until the next day once they know what the transformation efficiency is. I'd like to do this next time to save myself a lot of trouble.

Securing Plates in Incubator Taping the plates to the floor of the incubator is not a very good method. I taped my plates. I had 6 stacks of 5 plates, and two of the stacks came loose and shook everywhere. A lot of the wells in these plates were just completely empty later on. After this experience, I started wedging my plates stuck in a box and taping the box in the incubator. This seemed to work quite well.

Plate Manipulations and Biomek Every little manipulation to my 26 plates was taking forever. I started talking to Johnny, and he told me about the Biomek. We actually setup a program for me to use to do all of the manipulations to my plates (performing a 1/10 dilution after overnight culture, adding IPTG, resuspending pellet in lysozyme, adding lysozyme, adding DNAse/MgCl2, and adding EDTA). The one step I won't use the Biomek for is removing the supernatent after a centrifugation and before resuspending the pellet in lysis buffer since I don't want the Biomek to remove the pellet but I do want to remove as much liquid as possible. Even though I had condensed my 26 plates down to 2 plates, I still used the Biomek to do these manipulations this time to see how it would work. I had a few errors here and there, but I think overall this will be a very good method for me to use for future plates.

I think that basically covers everything I wanted to bring up.

1-10-12 Kathy and I met to discuss the topics

She felt that the most likely reason the transformation efficiency did not work as well when we did so many at once was because the DNA sat with the bacteria too long before electroporating. We shouldn't let the DNA sit for longer than 1 min since there could be things in the solution which degrade it. She felt that the delay about getting them into the incubator was not as big of an issue. We both agreed that it is possible that there was variation in the batches of commercial competent cells I had. She mentioned that I should do a PUC19 QC for new batches to make sure that the cells are good.

She agreed that I should transform, titrate, store the solution in the fridge, and then dilute and separate the culture the next day once I know what the transformation efficiency is.

She suggested that I might want to drill a hole in a cardboard box to secure it to the floor. This could be a good idea.

She is fine with me doing plate manipulations with the Biomek. Great!