Check if Enzyme died over break

krj439Enzyme Activity Assay Check 1_24_11Looks like it might have died. I used the new spectrophotometer and the cuvette wasn’t very full so I might retry with the other cuvette.

So long and thanks for all the…chocolates?

We finally made it!!!!!! ok so for my last week in lab I redid my DMSO assay and the results still showed an upward trend. Then I did 3 inhibition assays, the last two of which showed a slight inhibition. If ya want to check out all that stuff just go to my student folder. My computer is still freaking out and not letting me post pictures. Maybe one day I will get a computer that doesn’t hate me. So now I’m off to work on my lab report. good luck on your finals and see you guys on sunday 🙂

Last week is almost here!!!!

Hey guys, hope you had fun during break. So week before last I started the tester assays: enzyme, substrate, and DMSO varying. I did the analysis on them and all seemed well until I hit the DMSO. I went in and worked on that but unfortunately my graph points kept going up instead of down. So tomorrow I’m going to come in and try to redo it to see what is wrong. Hopefully the numbers will turn out better this time. My main componet for this week was doing more the analysis on VDS. I’m having a bit of trouble finding the Lipinski stuff for the NIH library. Any one know where I can find that?

Sorry I was late posting

Hey guys. IT’s been a crazy busy week with end of semester tests and papers. So I came in and did my assays that varied my enzyme, pNPP, and DMSO. My best enzyme amount was 30 uL, my best pNPP amount was 6ul. I came in yesterday and did the varying DMSO assay and I’m doing the charts and stuff on it today. I’ve already taken my plot points. But be careful if you are trying to do all 9 tubes prep work at once. I had a system going but sadly I was feeling to confidant and started going too fast. All I had left to add was DMSO before I headed over to the waterbath with the tubes and pNPP. I some how got flipped around and started putting the stuff for A,B,C, etc into the tubes for I,H,G, etc. I had to start all over 😦 But besides that I was able to work pretty efficiently. to make sure I have almost 10 minutes exactly of reaction in each tube, I would add pNPP and put a new tube in every 30 seconds. Then when the timer hit ten, I started pulling them out. Well that’s all for now. Hope you guys had a fun weekend!

Oh the top ligands is done!

My top ligands finally stopped running around 1Pm on Sunday. I didn’t have internet access until I got back from Galveston so I didn’t start on the Virtual stuff until Tuesday. I found the top 5 but My computerwas freezing when I was trying to find the top ligands. It finally worked after letting it sit for 10 minutes. GRRRRRR

I did protein characterization and concentration. So I now have my protein ready for assays so I’ll be doing that next week.

350mL later

so last week I did sonification and spun my protein down. Well since I thought I was suppposed to use the big tubes I added water so that it would be up to the 70-80% full line…which was a horrible idea. Needless to say, while others had only 10-20 mL to take through protein purification, I had to deal with 350 mL. What a mess! It took 2 days (about 5 hours total) to do a process that should have only take 1 hr. Now I’m trying to finish off my Top Ligands run but sadly it is running incredibly slow. Hopefully I’ll have internet access in Galveston so I can finish up analysis. Well see you guys next week! I’m off to enjoy my birthday weekend 🙂

Hoping for Success

So I came in on Sunday and started up my overnight transformation, growing up 2 colonies this time. Then on Monday I did the OD600 step which took from 8-11:30! (It was only supposed to take 2 hours max). So I ended up missing a class; oops. Then I let the IPTG do it’s magic. I came in 4 hours later and spun down my samples in the Beckman spinner. I switched the pellets over so that everyone else would have access to the tubes. I came in today and did the sonification step. Sadly all the tubes were full when I went to spin the stuff down. I ended up having to use the little spinner, but not until after I added a lot of water so I had to do two sets of spins. Next week I’ll be off to Protein purification!

A barely fruitful week

This week has been an odd mix of virtual screening and wet lab work. On the 19th I checked my virtual screening one and my 2nd attempt at the 1st run of H9 library was still going. Today I managed to start the second run of it. I also started the NIH clinical Library which is also now done. I began my analysis of the Cb 306 library and will hopefully be able to finish that one and the others’ analysis next week and upload some results. As for wet lab the only day that would have worked for me to do the long Protein expression scale up would have been today. I got the transformation ready last night and I talked to someone about getting more LB. Unfortunately as I was rushing out of lab, Dr B mentioned something about using another person’s stock of LB. The next morning I came in a checked the fridge and there was not nearly enough so I couldn’t continue. Turns out what he wanted me to do was use the LB that was sitting one the shelf area above the counters…so I guess LB doesn’t have to be refrigerated. It was too late in the day to continue so I ended up spending today making more LB (please don’t use it all! I need 1.2 L) and making an SDS page gel. I’ll be coming in on Sunday to prep things for a long lab day on Monday.

Bipolar week

So seems like the past couple week in lab have been one failure after another. My HL9 library virtual screeen had been running since the 5th when I looked at it yesterday. There were only 10953 ligands done. #9 was supposed to be running all 4 blades but it was sitting at .1% usage and #10 was supposed to be running 2 bladesd at it was at 23%. Dr. B had me kill it on #9 and start the cb_306 little library on it. Sadly #10 fell to .2% and this morning when i checked it the job seemed to have stopped completey. There was no pid fill and still only about 10953 ligand. Since my cb_306 library was done running, I restarted the other library from scratch. hopefully it works this time!

On to wet lab news: my wierd plates soon were joined by Yiling’s which also looked liquidy. Kathryn grew up some good plates using the ones she made so it’s not the plates that are screwed up. Also Yiling’s cells weren’t Bl21 but were Dh5alpha so that wasn’t the problem either. so it’ll remain a mystery what happened there. The 2 colonies i did get on the 2nd plate I made seem to have died because neither overnight transformation grew. Then my luck started to change. i grew up a new plate yesterday and it is beautiful. There are colonies galore so next week wish me luck!

It’s that time of the week again

krj_midi_analysisoct6

Wierd plates when I tried to grow up my colonies after transformation

So another week in the lab. OK I finally learned how to upload pictures. Yes I know, I’m a bit behind on the times. So last week when I made my plates they looked a little watery around the colonies. I put them in the fridge over the weekend and since they looked weird and did not give me the right results last week, I grew up a tube of each colony over the weekend. I came in Monday morning to find that all were clear except one tube with this weird mucus-looking gunk from colony 6. The camera was broke so I didn’t get to take a picture. Yesterday I re-looked at my weird plates and saw that the liquid kept growing so I’m thinking a fungus got into it. My new plates only had 2 colonies on the plate with 10 ul of bacteria in it. one of the colonies looks a little liquidy so I’m not sure if it is right either. I’m growing up a test overnight transformation that I’ll check tomorrow. The other thing I did this week was send my Midi-Prep DNA to DNA sequencing.Tthe DNA came out right so it should not cause problems on the plate. I also set up my first virtual screening.

Week of 9/27-10/1

So this week has had it’s ups and downs. On the bright side I have gotten my journal club article out of the way so that’s good. Hope you guys liked (or at least didn’t fall asleep during) the presentation. I know I wanted to 😛

Well on to the wet lab. I was pretty pumped because last week i got my protein to work but of course that fun luck can’t last so I’m back to the 90% failure part of lab. I started out by taking the pellets that I got last week from transforming my competant cells and getting them ready for Midi-prep. Kathryn has been really helping me out a lot this week so when 2/3 of my cell solution spilled, she helped me alter the protocol so that I could continue without having to redo about 4 hours of work so kudos to Kathryn! Unfortunately my results still came back with pretty low concentrations (about 20.4ng/ul). I defenitly have enough ng’s of DNA but it is too low of a concetration to send in for accurate DNA sequncing. I’m still going to try and send it in next week but I’m expecting a lot of N’s. In happy fun new, I’m sure everyone will be happy to hear that the new Midi-kit is much faster. Instead of the original 4 hours, it takes roughly 2 hours.

The last think I was doing this week was trying to follow the Protein Expression Scaled Up protocol. Lat week somehow I managed to grab the only tube in the BL21(DE3) box that was wrong, the control tube with no cells, so I had to restart this week. It seemed to be successful the 2nd time around so then I made a plate and let it grow up over night. I came in the next day and made a 50ml culture and let it grow up overnight. I came bright an early this morning expecting a long day, but unfortunately something went wrong and nothing grew up. It seemed like I was getting low concentrations at the beginning of the day so I let it sit but it still hasn’t grown so I’m just getting rid of it. Now I’m growing up 10 tubes with different colonies to check if maybe my colonies weren’t any good. So if you see 10 tubes in the shaker all weekend that’s what they are. Hopefully you guys have had a successful week and have a fun weekend. See ya next week!

Week of 9/20-9/24

Finally some success! On Monday morning a sent in Sample 3 and 4 (both of which came from the same colony) to DNA sequencing. #3 reflected the appearance of the other samples and well as looked like cut pNIC-Bsa4 so I assumed it had failed. #4 looked slightly different but still did not look like the online simulation of the cut Zhang gene. When I got the results back, I blasted the forward and reverse sample 4 with Zhang and it came out as a perfect match. This was much better than I expected. Then, I had to blast #3 because if it turned out to not be Zhang, I would have a mixed colony which could cause problems in the future. It also matched much to my surprise. Then I found the colony on my original plate that both samples came from and grew up 2 flasks of 80ml each of this colony. It grew over night and left me with large pellets after being spun down. Next week I’m going to send a sample to DNA sequencing again because my plate had overgrown a bit and the colony might have been tainted. Hopefully I will be moving forward  next week.

On a different note, if anyone can tell me how to embed images, I would really appreciate that. Hipe you all have a fun weekend!

Hours and hours later…

So i took those 3 colonies and ran with them. I took 2 samples of each of the colonies so that I would have 6 total samples. I miniprepped each one and now I am running an RE digest using the enzyme BsaI at 50 degrees for 2 hours and then 60 degrees for 20 min. Hopefully tomorrow I should be able to run the check gel.

PCR- it’s finally over…I hope.

KRJ_9/2/10 pNIC cloning gel

KRJ_8_31_10 pNIC cloning gel-1

The gel from 8/31/10 was Run 1 of PCR cloning for the Fall. The top bands are exactly what I’m looking for but unfortunately the bottom band seems to be some sort of contaminant. If it weren’t so dark, it might have been primer dimers but that is probably not the case. The 2nd Run on 9/2/10 showed much better results. The dark bands that represented  Zhang was present and the lower bands were significantly lighter or not even there. The procedures used were exactly the same (annealing temp of 60 degrees for 20 cycles) so something must have just been contaminated when it was used on the first run.