Another "harvesting cells day" for RNA isolation!

If everything went well, this will be my last "harvesting cells day". After today, I will have Rifr mutant cells that have grown in 0.05ug/ml of Rif and 0.025ug/ml of Rif with sBHI. I will also have my new Eryr mutant cells that have grown in 0.1ug/ml of Ery. Then the next step is to do RNA isolation --> cDNA synthesis --> cDNA labeling --> finally: Hybridization! (mutant vs wildtype!!). Hopefully I will get all the RNA isolation done by next week. I am officially moving to Richmond on Sept 13/14! so much packing.. so much junks..

Last Friday I streaked some cells ( the new strain, C1/RD) on to new sBHI, Ery4, Ery20, Ery40 & Ery64ug/ml plates. Today I checked the plates and found that the cells grew very well on sBHI and Ery4 plates, some growth on Ery20 plate but no growth on Ery40 and Ery64 plates. I am not sure what went wrong.. the strain should grow on Ery64 plate... anyways, going to redo this experiment later... maybe using overnight culture instead of cells from sBHI plate. Today going to streak some cells (my old OLD Rif mutant I got 2 years ago!) on to sBHI plates --> tomorrow pick 4 colonies and make some overnight cultures --> after tomorrow, harvest those cells for RNA isolation. such a nice day today! so sunny!

C1/Rd EryR strain

My "EryR" strain arrived yesterday after a long wait @ Fedex!! So I streaked some cells (kindly donated by Dr.Appelbaum's lab) onto sBHI plate and chocolate agar plate (kindly donated by Heather! *i wonder which undergrad blood was in it...*). I also made some overnight culture. Today: Checked my plates! they all grew!! YEHH! but it seems the strain grew better on sBHI than chocolate agar.. maybe it is more adapted to sBHI than chocoalte agar. Going to make some forzen stock today. I also streaked cells from my sBHI plates onto some Ery plates @ concentrate 4, 20, 40 and 64ug/ml. Let's hope they all grew tomorrow! Then I will start my RNA isolation next week. aww.. summer... just 2 more weeks then is back to school.

hum...

I don't think I will do the labeling today because I am not ready to do hybridization anytime sooner. I am scare the intensity of the probe will go down if I don't use it fast. So, I am going to wait for the mutant strain to arrive -> test its Ery resistant level ->then collect cells for RNA isolation ->then cDNA synthesis ->then do the labeling of both mutant and wildtype at the same time then I can do the hybridization! yup.. that is my plan for now!! Rosie: I saw Julian the other day when I was upstair doing cDNA synthesis. I told him about the strain and he said is great! so once I have tested its resistant level, I CAN USE IT! yahHH! Picture of the day: this time is the devil! kekeke

cDNA synthesis

(Yesterday) Finally did my very 1st cDNA synthesis of WT kw20! It was much easier than doing RNA isolation. Planning to do cDNA labeling tomorrow (my 1st time too! =P )

For the past month I have been trying to isolate a high-level erythromycin (Ery) resistance Rd strain by transformation with DNA from mutant strains provided by Dr.Appelbaum lab. But, unfortunately I have been unable to obtain such mutant strain. So I found another paper published by the same lab (Dr.Appelbaum) and found that they have successfully transformed the Rd strain to high-level Ery resistance (MIC 64ug/ml)some years ago. With the help of Rosie and Heather, I got a permit that allow strain to be imported across the border! yAh!! and I just got an email from them saying that they will send me the strain on Tuesday!!! yaHhHHhh!!!

Picture of the day:

Hiiiiiiiii