Team:UIUC Illinois/Notebook
From 2014.igem.org
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+ | <div class ="tbnote"> | ||
+ | <h2>Friday 15<sup>th</sup> August</h2> | ||
+ | <a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a> | ||
+ | <div style="clear: both;"></div> | ||
+ | <ul style="text-indent:30px;"> | ||
+ | <br><br> | ||
+ | Pick 1-8 big colony | ||
+ | <li>4-10 middle colony </li> | ||
+ | <li>17-24 small colony to 50uL LB</li> | ||
+ | <br><br> | ||
+ | Set up 24 10uL PCR reactions | ||
+ | <li>260uL PCR</li> | ||
+ | <li>52mL buffer</li> | ||
+ | <li>5.2uL dNTPs</li> | ||
+ | <li>0.5uL for ndmB</li> | ||
+ | <li>1.5uL rev ndmB</li> | ||
+ | <li>2.5uL polymerase</li> | ||
+ | <li>180uL H2O</li> | ||
+ | |||
+ | |||
+ | |||
+ | </ul> | ||
+ | </div> | ||
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+ | </html> | ||
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+ | <html><div id="Monday_18th_August.html"></div></html> | ||
+ | <html> | ||
+ | <div class ="tbnote"> | ||
+ | <h2>Monday 18<sup>th</sup> August</h2> | ||
+ | <a href="https://2014.igem.org/Team:UIUC_Illinois/Project/Overview" target="_blank" class="tbnotelogo PSlogo"> ASDF </a> | ||
+ | <div style="clear: both;"></div> | ||
+ | <ul style="text-indent:30px;"> | ||
+ | <br><br> | ||
+ | Inoculate 23 and 24 (PCR) in 5mL LB | ||
+ | <br><br> | ||
+ | Inoculate IG4/IG5 in 5mL LB | ||
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+ | |||
+ | |||
+ | </ul> | ||
+ | </div> | ||
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Revision as of 23:19, 17 October 2014
-
PROJECT NOTES
- NOTES BY MONTH
Monday 9th June
ASDF Made LB broth using 10 grams of bacto agar + 12 grams HiVeg LB broth + 600 mL ddH2OAutoclaved 1.5 mL Eppendorf tubes as well as LB
Poured plates of LB and chloramphenicol (CAM)
Streaked 2 plates with E. coli DguaB (pDCAF3)
Incubated in 37 degrees celsius
Aliquoted kanamycin (KAN) in four 1mL tubes
Tuesday 10th June
ASDF Autoclaved at 1:30pm- 0.8 grams of YNB + 156 mL H2O
- 28 grams of salts + 490 mL H2O
Used sterile filter for 1M MgSO4 and 1M CaCl2
Suspended 5 uL of CM34 (antibiotic) + 5 mL of LB + 1 colony of pDCAF3 E. coli
Added .5 grams of caffeine + 400 uL of 1M MgSO4 + 20 uL 1M CaCl2 + 40 mL M95x salts + H2O to YNB + H2O mixture
- Labeled this "M9 media"
Inoculated frozen stock of E. coli guaB pDCAF3 in 5 mL of M9 media
Placed sample in 30 degree celsius shaker for culturing
Wednesday 11th June
ASDF Inoculated CBB1 frozen stock in 5 mL of M9 mediaPlaced sample in 30 degree celsius shaker
Purified pDCAF plasmid using DNA mini kit
- Plasmid concentration was 132.9 ng/uL
Friday 13th June
ASDF Added 10 uL deionized H2O into the J23114 promoter plasmid (Ampᴿ)Followed bacterial transformation protocol to prepare/grow culture overnight
Inoculated 5 mL of M9 with CBB1 and placed it in 30 degree celsius shaker for 3 days
- Previous CBB1 culture did not grow, showed slight growth
- pDCAF3 strain in M9 did grow in the 30 degree celsius shaker
Monday 16th June
ASDF No growth in M9 by CBB1To grow the promoter plasmid:
- 5 mL of LB + 1 uL Amp
- Label tubes
- Grow in 30 degree celsius shaker overnight
- 2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample
Purify genomic DNA plasmid following the Wizard Protocol
- Added lysis buffer before centrifuging. So we separated (translate) sample into 2 tubes and will (translate) accordingly
- We put our 2 promoter plasmid plates in the 4 degree celsius shelf
Tuesday 17th June
ASDF Quantified gDNA with TECAN- Result: 5.4 ng/uL
Made 2 plates of M9 culture and 2 plates of CBB1 culture (100 uL used) then placed them in 37 degree celsius shaker
Wednesday 18th June
ASDF Autoclaved 50% glycerolMade four 25% glycerol stock solutions (stored in the -80 degree celsius freezer):
- CBB1 in M9 (x2 1.5 mL tubes)
- Promoter 1
- Promoter 2 (both promoter plasmids are J23114 Ampᴿ)
- Stored in the -20 degree celsius freezer in the DNA box
Thursday 19th June
ASDF Quantified promoter plasmid (J23114 #1 and #2)-
#1: 342.9 ng/ul with a ratio of 1.9
#2: 373.2 ng/ul with a ratio of 1.91
Checking to see if expired enzymes work: Using the J23114 promoter plasmid, we will see if the 3-year expired SpeI works
- We combined 1 uL of 2.1 buffer, 1 uL of Spe1, and 6 uL of J23114 promoter
- Heat bath at 37 degrees celsius for 1 hour because 2.1 buffer is <100% effective on this enzyme
- Then we will run gel electrophoresis: Concluded that the enzyme works
Cutting the promoter with restriction enzymes
- Combined 24 uL of J23114 promoter + 3 uL of CutSmart buffer + 1 uL of each EcoRI and SpeI
- Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius
Cutting linearized backbone
- Linearized backbone with chloraphenicol marker (12 uL) + 12 uL water + 1 uL of each EcoRI, PstI, and DpnI + 3 uL 2.1 buffer
- Heat bath at 37 degrees celsius for 1 hour + 20 minute heat-kill at 80 degrees celsius
Wednesday 9th July
ASDF Made 400mL of M9 solution- Made 40% glucose solution of 50mL: 2g glucose
- Added the following to 316mL of DI H2O: Na2HPO4: 13.56g Na2HPO4, 2g NH4Cl, 1g NaCl, 6g KH2PO4
- Added 80mL 5x M9 salt solution (20% of 400mL)
- Added 800uL MgSO4 + 40uL CaCl2
- Added 4mL glucose solution using syringe
- Stirred until all dissolved
Removed competent cells from the -80 freezer
Thawed on ice for 30 minutes
Removed CAM agar plate from 4 degree cold room
Mixed 4uL of pDCAF3 plasmid with 30uL competent cells in microcentrifuge tube, placed on ice for 30 minutes
Placed mixture in water bath (42C) for 45 seconds
Placed mixture on ice for 2 minutes
Added 300uL LB to mixture
Placed in 37 degree shaker for 45 minutes
Plated cells on CAM plate, placed in 37 degree drawer overnight
Thursday 10th July
ASDF GuaB/pDCAF suspension in caffeine/theobromine- 0.0975g/10mL caffeine, 0.09g/10mL -> 50 mM
- 2.5mL of M9 (with Glucose) + 2.4875mL of H2O + 12.5uL of caffeine/theobromine
- Use syringe to filter out 10mL of caffeine/theobromine into new conical test tubes
- Take out guaB/pDCAF from -80C freezer, suspend it using 10uL pipette tips
- Place them into 37C inclubator overnight
Wednesday 16th July
ASDF Incubate 4 tubes with LB+CM- Red 1 and 2
- Green 1 and 2
- Placed in shaker at 12:20pm
Thursday 17th July
ASDF Made 1mg/mL Erm of 50mLUsed syringe to filter out
25mL Erm into LB agar
Made 13 places
Erm is in -20C
Friday 18th July
ASDF Streak out Erm-R onto Erm plates (made 2)Put them into incubator (start: 9:35am)
Monday 21th July
ASDF cdh PCR redo: Made 4 master mixes: 5uL gDNA CBB1, 45uL DDH2O Master Mix 1: Regular-
20uL Buffer G
4uL forward primer
4uL reverse primer
0.4uL Q5 polymerase
2uL template
9.6uL H2O
Saturday 26th July
ASDF MRS agar of 200mL- 3g agar (15g/L)
- 10.41g MRS
300mL LB + 3uL CM34 + IG4
300mL LB + 3uL CM34 + IG5
Monday 28th July
ASDF Diluted caffeine and theobromine solutions for HPLC- 500uM Caffeine (10mL)
- 500uM Theobromine (10mL)
- 100uM Caffeine (10mL)
- 100uM Theobromine (10mL)
- 250uM Caffeine + 250uM Theobromine (10mL)
(page 169 on bottom, can’t read XW’s part)
- Take 1mL of I4/I5 LB cultures and spin for 2 minutes at 8000 rpm
- Removed supernatant
- Added 1mL of OH salt, resuspended
- 2 minutes at 8000 rpm
- Removed supernatant
- Resuspended in 1mL of M9 media
Thursday 31th July
ASDF gDNA qualtify: 97.5 ng/mL, 1.76 ratiocdh PCR
- 30uL H20
- 10uL Phusion HF
- 1uL dNTP 10x
- 2.5uL rev.
- 2.5uL for.
- 3.5uL template
- 0.5uL polymerase
- Made 4 10uL samples from master mix
- Gradient: 44.9, 50.8, 58.9, 65.1C
- Cycle 3 was used
- pdCAF3
- .5uL Cutsmart buffer
- .5uL Saci
- .5uL EcoRI-HF
- 7.52uL pdCAF3
- 36.48uL H2O
- Green .5uL Cutsmart buffer .5uL NcoI .5uL NheI 8.4uL Green optogene 35.1 uL H2O
- Incubated at 37C waterbath for 1 minute
- Gel run at 120V for 25 minutes with a 1kb ladder
- 3mL M9, 300uL Caffeine, for only IG4, 30uL cells
- Placed in 37 degree shaker
Performed restriction digestion of pdCAF3 and green optogene
Cultured IG4/IG5 in M9 and no CM
Monday 4th July
ASDF M9 Caffeine Media- 200uM 200mL caffeine
- 50 M9 5x salts
- .5mL MgSO4
- .025mL CaCl2
- 1.25mL 40% glucose
- 200mL 200uM Theobromine
- 50mL M9 5x salts
- .5mL MgSO4
- .025mL CaCl2
- 1.25mL 40% glucose
- 5uL cutsmart buffer
- 1uL PstI
- .5uL XhoI
- 7.52uL pdCAF3
- 35.98uL H2O
- 1 hour 37C incubation
- 20 min 80C heat inactivation
M9-Theobromine Media
pdCAF3 Digestion
Monday 11th August
ASDF-
Resuspended primers in water
- Spin down for 5 seconds
- Inverted several times, set for 3 minutes
- Inverte again, let sit for 3 more minutes
- Invert before use
- 57uL H2O + 1.6uL dNTP + 16uL buffer + 0.8uL polymerase in tube 1
- 9uL of 1 + 0.25uL BB primer for + 0.25uL primer rev + 0.5uL template (PSBC13) in tube 8
- Add 0.5uL pdCAF3 to tube 1
- 9uL of 1 to tube 2-7, 0.25 rev + 0.25 for to each of the tubes respectively
- Spin down for 5 seconds
- Put tube 2-8 in PCR machine
PCR
Tuesday 12th August
ASDF-
456uL H2O, 128uL buffer, 12.8uL dNTP, 6.4uL polymerase into tube A
9uL of A + 0.25uL BB for + 0.25uL BB rev + 0.5uL purified green in tube 8
0.5uL pdCAF to tube A
72uL A to tube 1-6, 9uL A to tube 7, 0.25 BB rev/for to 7, 2uL for/2uL rev to 1-6 respectively
Spin down for 5 seconds
PCR in cycle 1
Tube | Primers | Template | Volume(uL) |
---|---|---|---|
1 | ndmA 1 | pdCAF | 80 |
2 | ndmA 2 | pdCAF | 80 |
3 | ndmB 1 | pdCAF | 80 |
4 | ndmB 2 | pdCAF | 80 |
5 | ndmC | pdCAF | 80 |
6 | ndmD + gst9 | pdCAF | 80 |
7 | BB | pdCAF | 10 |
8 | BB | Green Optogene | 10 |
Wednesday 13th August
ASDF- Add 114uL H2O, 32uL buffer, 3.2uL dNTP, 1.6uL polymerase to tube 1
- 72uL of A to tube 10 + 20uL BB for + 2uL BB rev + 0.5uL purified green
- 72uL of A to tube A + 2uL ndmD + gst9 + 2uL for/rev
- Spin for 5 seconds
- Put in PCR machine
- Cut one gel piece as small as possible
- Add 500uL of buffer QG to tube 1-4, 10, 11, incubate at 50C for 10 minutes - invert several times until dissolved
- Load liquid in column, spin 1 minute at full speed
- Discard flow through, add 500uL QG, spin 1 minute at full speed
- Discard flow through, add 750uL DNA wash buffer, spin 1 minute at full speed
- Discard flow through, spin 1 minute at full speed
- Discard flow through, add 40uL H2O, move column to fresh tube, spin 1 minute at full speed, sit for 1 minute
PCR
Gel Purification
Tube | ng/uL | H2O(uL) | uL |
---|---|---|---|
ndmA 1 | 55.8 | N/A | 0,448 |
ndmA 2 | 64 | 14 | 0.55 |
ndmB 1 | 37.9 | 12 | 0.79 |
ndmB 2 | 55.4 | 4 | 0.9 |
ndmC | 41.9 | N/A | 0.6 |
ndmD/9 | 85.6 | N/A | 0.6 |
BB | 2.5 | 20 | 0.76 |
Thursday 14th August
ASDF- Throw Top 10 cells on ice for 10-20 minutes
- Add 5uL of GG rxn mix then stir it gently on pipette
- Leave cells on ice for 20-30 minutes
- Heat shock at 42C for 5 minutes
- Put 1mL LB into the tube, mix it, then transfer it and leave it in 37C for 1 hour
- Plate 20uL and 200uL on LB+CM
Ran gel
Transformation
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Friday 15th August
ASDF- 4-10 middle colony
- 17-24 small colony to 50uL LB
- 260uL PCR
- 52mL buffer
- 5.2uL dNTPs
- 0.5uL for ndmB
- 1.5uL rev ndmB
- 2.5uL polymerase
- 180uL H2O
Pick 1-8 big colony
Set up 24 10uL PCR reactions
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