Team:UIUC Illinois/Notebook
From 2014.igem.org
(Difference between revisions)
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<li>Label tubes</li> | <li>Label tubes</li> | ||
<li>Grow in 30 degree celsius shaker overnight</li> | <li>Grow in 30 degree celsius shaker overnight</li> | ||
- | </ul | + | </ul> |
New CBB1 trials:<br><br> | New CBB1 trials:<br><br> | ||
<ul style="text-indent:30px;"><li>2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample</li></ul><br><br> | <ul style="text-indent:30px;"><li>2 tubes of 1 mL M9 + 2 tubes of 1 mL LB each with 10 uL of sample</li></ul><br><br> | ||
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<li>Promoter 1 </li> | <li>Promoter 1 </li> | ||
<li>Promoter 2 (both promoter plasmids are J23114 Ampᴿ)</li> | <li>Promoter 2 (both promoter plasmids are J23114 Ampᴿ)</li> | ||
- | </ul | + | </ul> |
- | Purified both promoter plasmids using the purification protocol | + | Purified both promoter plasmids using the purification protocol<br> |
<ul style="text-indent:30px;"><li>Stored in the -20 degree celsius freezer in the DNA box</li></ul><br><br> | <ul style="text-indent:30px;"><li>Stored in the -20 degree celsius freezer in the DNA box</li></ul><br><br> | ||
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Revision as of 02:02, 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