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Template:Team:UC Berkeley/Notebook/CDB notes
From 2008.igem.org
6/4/08 - Today we continued training and discussed the project.
6/6/08 - Jin streaked a plate with MC1061 E. coli cells and incubated at 37 C.
6/7/08 - Plate moved to fridge.
6/8/08 - I chose an isolated colony from the plate and inoculated a few mL of fresh LB media with it. The tube was incubated on a shaker at 37 C for 24 hrs.
6/9/08 - Took 200 uL of saturated culture and added it to 8 mL of LB media at 10:45 AM. Grew for 2 hrs on a shaker at 37 C.
Split culture into 3 tubes with 2 mL each. Culture 1 - Control (no sound), grew in foam box. Culture 2 - Music, high volume for 1 hr. Culture 3 - Ultrasound bath for 1 hr.
Note - Didn't have lysozyme thawed for RNA purification. Will repeat experiment tomorrow.
Inoculated a new tube of media with an isolated MC1061 colony.
6/10/08 - Added 200 uL of saturated culture to 8 mL of LB media. Grew for 2 hrs on a shaker at 37 C.
Split culture into 3 tubes with 2 mL each. Culture 1 - Control (no sound), grew in foam box. Culture 2 - Music, high volume for 1/2 hr. Culture 3 - Ultrasound bath for 1/2 hr.
Performed RNA extraction with RNA protect kit for bacteria -
Protocol
Supplies Sterile, RNase-free pipet tips
Suitably sized tubes and microcentrifuge or centrifuge with appropriate rotors
Lysozyme (e.g., Sigma, cat. no. L7651) or appropriate lytic enzyme*
Tris and EDTA for preparing TE buffer
QIAGEN Proteinase K (see ordering information, page 47)
2 ml Safe-Lock tubes (Eppendorf, cat. no. 0030 120.094)*
14.3 M b-mercaptoethanol (b-ME) (stock solutions are usually 14.3 M)
RNeasy Protect Bacteria Mini Kit
Ethanol (96–100%), ethanol (80%), or ethanol (70%)†
Optional: RNase-Free DNase Set (see ordering information, page 47)
RNA zap
100 ml lysozyme-containing TE buffer (10 mg lysozyme in 25 ml RNase-free TE)
DEPC water
RNASE-free TE
beta mercaptoethanol
200 proof ethanol
RNase free needle and syringe
Before starting -
clean work area, gather all pipets and tips
mix Buffer RLT and beta mercaptoethanol (10 uL BME per 1 mL RLT) - need 20 uL BME in 2 mL RLT
mix TE Buffer with lysozyme (15 mg/mL) – need 7.5 mg lysosyme in 500 uL TE Buffer
dilute buffer RPE 1:4 with ethanol - need 44 mL ethanol
prepare DNase stock solution - dissolve solid DNase in 550 uL of RNase free water using an RNase free needle and syringe. Mix carefully with needle - DON'T centrifuge. Freeze single-use aliquots at -20C
After Sound Treatment
Protocol 4
in a 2 mL tube, add 1 mL RNA protect and 500 uL culture. Vortex 5 s and incubate 5 min (RT). Centrifuge for 10 min at 5000xg. Decant supernat. Dab inverted tube once on a paper towel - pellet may not be visible
Mix 15 uL Proteinase K with 100 uL TE Buffer. Add mixture to pellet and carefully resuspend the pellet by pipeting. Vortex for 10 s. Incubate for 10 min (RT) on a shaker (or vortex 10 s every 2 min) - extending incubation time may increase yield.
Add 350 uL of Buffer RLT and vortex vigorously- if there is particulate matter, centrifuge 2 min at > 8000 x g and use only supernatant
Add 200 uL of 96-100% ethanol and mix by pipetting (DON'T centrifuge)
Protocol 7
Transfer up to 700 uL of lysate, inc any ppt to RNeasy mini spin column in a 2 mL collection tube. Centrifuge for 15 s at max speed. Discard flow-through.
Repeat if there is remaining lysate
Appendix B - DNase digestion
Add 350 uL of Buffer RW1 to mini spin column. Centrifuge for 15 s at max speed. Discard flow-through
Add 10 uL of DNase 1 stock sln to 70 uL of Buffer RDD. Mix by inverting gently - don't vortex. Centrifuge briefly
Add DNase 1 mixture (80 uL) directly to mini spin column membrane and incubate (RT) for 15 min
Add 350 uL of Buffer RW1 to spin column. Wait 5 min, then centrifuge for 15 s at max speed. Discard flow through and collection tube
Protocol 7 cont.
Place spin column in a new 2 mL collection tube.
Add 500 uL of Buffer RPE to spin column. Centrifuge for 15 s at max speed. Discard flow-through
Add 500 uL of Buffer RPE. Centrifuge for 2 min at > 8000 x g. Carefully remove spin column from collection tube and discard tube and flow through.
optional - Place spin column in a new 2 ml tube and centrifuge for 1 min. Discard tube and flow through.
Place spin column in a new 1.5 mL collection tube.
Add 50 uL of RNase free water to spin column membrane. Centrifuge for 1 min at > 8000 x g to elute RNA
If expected yield is over 30 ug repeat last step. Add 30-50 uL of RNase free water to spin column membrane. Centrifuge for 1 min at > 8000 x g to elute RNA
Discard spin column and put tube on ice
Results
Sonic Bath - 5.1 ug/mL 260/280 - 1.61
Music - 1.4 ug/mL 260/280 - 1.72
Control - 4.2 ug/mL 260/280 - 1.67
Since purity is low, experiment was repeated with the following changes:
1) assumed a higher # of cells - increased TE Buffer to 200uL, Buffer RLT to 700 uL and ethanol to 500 uL.
2) centrifuged after addition of buffer RLT and used only supernatant.
3) eluted with 100 uL of RNase-free water (2 additions of 50 uL each) - Note - The concentration of RNA after the addition of 100 uL of water was substantially higher than the concentrations in any of the first samples, which were only eluted with 50 uL. Not sure why... but it's good because it means we would have enough total RNA for the microarray.
4) Centrifuged at max speed, except for the initial pellet.
Much better results this time 260/280 was 2.01 and the concentration was 20.6 ug/mL.
I inoculated some media and will try again tomorrow.
- Updated protocol here [http://docs.google.com/Doc?docid=ddh89z4j_11cnxq6jwr&hl=en]
6-11-08
Repeated updated protocol from 6/10/08
Results
Sonic Bath - 18.2 ug/mL 260/280 - 2.04
Music - 11.6 ug/mL 260/280 - 2.06
Control - 20.5 ug/mL 260/280 - 2.04
Used 10 uL to test the purity and concentration using a spectrophotometer. Don't have enough total RNA from the control sample for the microarray. Need to repeat control experiment to extract enough RNA. Need to concentrate the music and sonic bath samples to 0.5 ug/uL (use zymo clean-up reaction.)
6-12-08
Tried to concentrate sonic and music samples using a Zymo DNA kit. Failed miserably! Both purity and concentration dropped. Need an RNA zymo kit.
Repeated experiment for all three conditions. Repeated protocol from 6-10-08, except used 150 uL collected in 3 sample tubes for each condition.
Spectrophotometer results here [http://spreadsheets.google.com/ccc?key=pWg-yF6Hhkf_1ytn5MB1sWA&hl=en]
Tomorrow, will concentrate RNA and run on a gel.
6-13-08
Used the microcon centrifugal filter devices ultracel YM-10 to concentrate RNA. Used 100 uL of sound sample and 150 uL of control and ultrasound sample. (Note: 1 of the 3 sound samples initially showed a low purity and was not used, hence only 100 uL of this sample). Used 50 uL of RNase free water to clear glycerol from tube before use. Spun at 14000 xg for 9 minutes (8 minutes for the sound sample). Flip over tube and run for 3 min at 1000xg to elute. The volume was reduced to approximately 60 uL of each sample. All samples show a 260/280 ratio of 2.01-2.02 (pure).
Final concentrations and purity
Sonic Bath - 0.735 ug/uL 260/280 - 2.02
Music - 0.8 ug/uL 260/280 - 2.01
Control - 0.52 ug/uL 260/280 - 2.01
Loaded 1 ug of each sample and ran a 1.2% e-gel. Two distinct bands appear for each sample (23S and 16S subunits).
6-16-08
Submitted form, 3 samples and 3 affymetrx microarray chips to Justin at 255 LSA. He will contact Chris when chips have been analyzed.
6-23-08
Didn't have much to do last week. Helped the others with gels, digestion, mini-prep and mixing buffers. Did some research on holin-antiholin. Downloaded GCOS (Gene chip operating software)- freeware from affemetrix to analyze microarrays. Played with some sample data so that I'm ready for microarray analysis when the time comes.
There was a delay with the FGL, so I won't get data for a few more days. Trying to keep myself from yelling at Justin (the microarray guy) for not telling us about the delay until today.
Began researching papers to create a model for the kinetics of the holin-antiholin-lysozyme system. Will model using a system of linear equations. It will be a more abstract model (not based on experimental data, which would be hard to measure). Need a system of equations for each of three conditions (before depolarization, after depolarization, after enough holes form to allow lysozyme to cross IM).
6-27-08
Still no microarray data. Justin from FGL hasn't ordered the reagents yet. Met with Karen Vranizan, the stats woman from FGL. She advised me not to use the GCOS program because there is no easy way to normalize samples across multiple chips. She recommended RMA Express, a freeware program that will normalize our data. I downloaded the program and began to learn how to use it so when I actually get data, I can analyze it.
I created construction files for three ultrasound-responsive promoters for the grpE, recA, fabA genes based on primers from the following papers
recA - primers for RecA promoter from Vollmer et al., 1997. [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=168554&blobtype=pdf]
fabA - Primers for fabA proteins from Bocher, et. al 2002
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-44W2K4N-6&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=b221542b03abe204b6638860617ae471]
Want to make a fourth promoter for katG, but it has four internal restriction sites, so I'm not sure if its worth ordering ten oligos. Also, I don't know how to get make a mutation "silent" in a noncoding region. Will ask for Jin's advice when he comes in.
