3ggggggggggggi3

1st experiment in 2007!!

2007-01-05T14:54:00.000-08:00

This experiment is related to Rosie's post: "Rachael's boyfriend's plasmid, and Rifampicin"

Back in the year..2005 I have already selected Rif resistant kw20 mutants. As I recall mutant "1" & "i" have only a one point mutation and mutant "R1" & "R2" have two points mutation (confirmed by the Davies' lab) but resist to a higher concentration of Rif than 1 & i.

So here's the plan discussed with Rosie:
Make some overnight culture of 1 or i and R1 or R2 and wildtype kw20 (control) -> take 100ul into 20ml of sBHI -> grow until O.D. = 1.0 -> take 1ml from the flask into a test tube with Map7 DNA -> roll for 15' -> DNase 5' -> series of dilution -> plate on Nov plates sBHI plates.

Result: Compare the TF between RifR mutants & wildtype kw20
When to do this? some time in Jan!

Let me know if I should make change to this plan above. Thanks!!!

cDNA synthesis.. a long LONG day..!

2006-11-15T18:02:00.000-08:00

well, I was doing cDNA synthesis (18samples) today.. and that require Reverse Transciptase ($185!!! for only 50ul = ~26samples). So I was adding RT to my samples and it ran out when I still have 4 samples left. I thought "there should be more right?? if not, the other lab should have it..." Then end up neither labs have it and I have to go to class (I was pulling my hair out when no one has RT but Andrew saved the day by helping me again!!! Thanks!!) so I just ran the 14 samples PCR before class (2hrs) and left the rest in the ice bucket until my classes were over. After classes, I went to Wesb to buy the RT and got free chocolate! Which cost way less than the 50ul of RT. So end up I spent more time than I planned :-(

I will be doing cDNA synthesis tomorrow again but I think tomorrow it will run more smoothly than today forsure!!!

I can believe I will be using ~$200 by the end of tomorrow!! I could use that money to buy something else..haha.. but technically that's not my $$$ ! kekeke Anyways, thanks Rosie for the support!~

Pretty Gel!

2006-11-08T17:23:00.000-08:00

Today I ran a 1% agarose gel with my 9 samples of RNA that I isolated in the past week or so. It was great! I just can't believe the gel is sooooOoOo nice and pretty! I am just glad all the samples look great. Big thanks to everyone in the lab, especially Andrew who gave me some of his 50X TAE and loading dye to use.

Now that I know my RNA is good, I can go on to cDNA synthesis (next week) and plan out what I am going to do for the next few months. I was scare before that my RNA samples might be bad so I didn't want to promise anything until I know the quality of my RNA.

Time to go back to my homework....

MORE RNA!!!! & lab meeting

2006-11-06T20:31:00.000-08:00

Spent another 7ish hrs doing RNA isolation again but this time I did 6 samples instead of 3. Since I have done RNA isolation twice already, I figured out I should be able to handle 6 samples and save some time for other things. Doing 3 samples more took about an extra hour of work but really.. I saved 5 hrs of work if I were to do 3 samples at a time. The bad things about doing 6 samples at a time are my hands get really tired from pipetting (recovering the upper phase step) and I need to be super careful not to mix the samples up. I am just glad is all over... hopefully.. just need to run a gel to check.. if everything look great then today will be my last day of doing RNA isolation!!! (at least in my last undergrad year..hahaha..)

As for lab meeting that just ended: It was great! Wasn't as bad as I thought... What was new about this time was I used diagram to explain my (most of) experiments and that seemed to work pretty well so I will continue to use diagram for my next lab meeting. I hope you all liked the weird food I brought today!

RNA RNA RNA RNA!! (yesterday)

2006-11-03T10:41:00.000-08:00

Took me ~6 hours to get some RNA sample... and 2 out of the 3 samples didn't yield as much RNA as I expected. (sample#1: 568.8ng/ul, #2: 976ng/ul & #3: 3421.1ng/ul). So I probably have to concentrate sample #1 & 2 before I can do cDNA synthesis. I should also run a gel next week just to be sure the RNA sample is good...

Just ~1 month before final exams start! Hope to get more done before that... I guess I will stay at the lab during X'mas too. (the undergrad will take over the LAB!! muhahahahahaha )

Lab meeting

2006-11-01T15:38:00.000-08:00

After Rosie's lab meeting yesterday.. I am still thinking about why deletion/addition mutation doesn't matter? Is it because eventually after many generations the mutation will cancel each other out (the number of deletion = the number of addition)? or is it sufficient enough to just have substitution mutation alone in the PERL simulation?

Anyhow, I will be presenting next week lab meeting... I don't even remember when was the last time I presented?? Maybe eariler this year?? I still need to think about what results to include and what not.. the presentation will include all the things I have done for the last ~1.5years. I still can't believe it took so long to isolate/find a erythromycin resistant H.influ mutant because it only took about 5 to 6 months to isolate a rifampicin resistant H.influ mutant. So the lab meeting next week will be about what I did to isolate/find this rare eryR mutant!!

What yummylicious food should I bring to lab meeting??? How about some Halloween candy/chocolate? hahaha just kidding.. I will bring something good.. hopefully... :P

Midterms...!

2006-10-16T13:59:00.000-07:00

due to mid-terms.. essays... presentation...cold... I was unable to take time to do some exciting experiments. Well.. I can take time to do them but I figured out that if I do my experiments hurryingly, my results might be bad bad bad.. then I have to re-do them again..etc So it is better if I get all my exams and homework done then do some experiments with great results!

Since I don't have experimental data to post.. I'll post something interesting I learned from my neuroscience class!

Botulinum toxin:
A type of botulinum toxin (BOTOX), "A", is used for the beauty treatment for facial lines and wrinkles.

How it works?
The toxin cleave the "snare" proteins needed to have vesicle fusion to occur. So without the fusion, no neurotransimitters are released --> no action potential --> muscles don't move

Good effects?
Your muscles don't move so the wrinkles seem to be gone.

Side effects?
Muscle degeneration! So with continuous use of Botox, your facial muscles will eventually be gone and more Botox will not help with the wrinkles anymore because you don't have any facial muscles left. (paraphrased from my prof)

Conclusion:
BOTOX is bad! We should just be who we are... :-)

awww...! B-Day party!

2006-09-21T13:32:00.000-07:00

Thanks everyone for the yummylicous cake + the pretty tiara (Queen of the Undergrads)!! I guess I can't call the "oldies" mean now (maybe just today)!

The search for Ery Resistant Mutant Strain officially ended this week!! What a relief... ! You guys can hear allll about it when it is my turn to present at lab meeting.

Finally!

2006-09-18T18:24:00.000-07:00

I am sure all of you are excited to hear about the *undergrad's* result for the past week!

So here it is..

My Ery Mutant cells (C1/RD) is actually just resistant to Ery 15ug/ml and not 64ug/ml as stated in the source lab's paper. They selected the cells on Ery 15ug/ml but they probably tested the cell's resistant in another way that gave them the result. Anyhow, I am happy with this already! I have done all I can do for the past year to find this mutant!! yehhh!!

So now.. all I need to do is concentrate on my microarray experiment! how exciting is that??? I just hope I will have time to do it with all those essays, MTs and homeworks waiting for me!! (not to mention the time being torture by the *oldies* in the lab) Muhahahaha~

The Stupid Undergrad!!!

2006-09-12T20:05:00.000-07:00

I did not grow my EryR cells with antibotics in the medium!! So maybe that is why my cells won't grow on 64ug/ml of Ery plate!

(Yesterday)Anyhow... luckily, I didn't throw out the chocolate agar slat I got from the source lab and I streak some more from that tube onto some freshly made plates (sBHI, Ery4, 10, and 20ug/ml plate)

Today: Surprisedly I can see super tiny colonies on the sBHI plate! I can't believe the cells are still alive in that tube because it was from a LONG TIME ago! I didn't see any colonies on any of the Ery plates so I leave the plates in the incubator and hope I will see some colonies tomorrow (Thanks Andrew for the suggestion that I should leave my plates 2 days in the incubator as it might take the cells longer time to *edit* Grow in the presence of antibiotics!).

PLEASE PLEASE PLEASE LET ME SEE SOME COLONIES TOMORROW!!!!!!

Testing C1/Rd strain again!

2006-09-06T19:20:00.000-07:00

I got my result for testing the resistance level of Ery of this new strain, C1/Rd, that I got from the other lab. On the paper it says after they transformed a Rd strain with their mutant DNA (C1), the new strain (C1/Rd) has a MIC of 64ug/ml. For some strange reasons, my new result tells me they can't even grow on 20ug/ml of Ery sBHI plate. So here is what I did -> I plated 100ul of overnight culture on each plate with Ery concentration of 0, 4, 20, 40 and 64ug/ml. The cells only grew well on 0 and 4ug/ml Ery plate.

Some possible reasons:
1. My plates are about 2.5weeks old (maybe I should make some new one!)
2. Some of my overnight cells died?
3. Any suggestions?? Should I email the person who kindly sent me the strain for help??

I think I will make some more new plates with Ery concentration of 0, 10, 20 and 40ug/ml. Then plate 100ul of overnight culture on them.

Suggestions are WELCOME!!

Another "harvesting cells day" for RNA isolation!

2006-08-30T09:09:00.000-07:00

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..

2006-08-21T13:25:00.000-07:00

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

2006-08-18T10:37:00.000-07:00

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...

2006-08-11T12:00:00.000-07:00

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

2006-08-10T13:48:00.000-07:00

(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

2006-08-09T12:36:00.000-07:00