Transformant Results
Plate | # of colonies |
1 | no colonies |
3 | 7 colonies |
4 | 19 colony |
pBAD | TMTC colonies |
|
Colony PCR
Protocol: See common protocol
- Changes: use Phusion pol; add MgCl2 and DMSO
- Samples to be PCRed:
- 2 from pBAD
- 3 from plate 3
- 4 from plate 4
- 1 H2O control
- Total: 10 samples + 1 extra = 11 samples
PCR Master mix
Reagent | 1x rxn volume (uL) | Master Mix |
5x rxn buffer | 5 | x11 | 55 |
10mM dNTP | 2 | x11 | 22 |
sdH2O | 10.55 | x11 | 116.05 |
Phusion polymerase | 0.2 | x11 | 2.2 |
MgCl2 | 2 | x11 | 22 |
DMSO - 5% | 1.25 | x11 | 13.75 |
10uM fw primer (G1004) | 2 | x11 | 22 |
10uM re primer (G1005) | 2 | x11 | 22 |
Total | 25 | | 275 |
PCR Tubes
From plate pBAD: | pT1 | pT2 |
From plate 3: | 3T1 | 3T2 | 3T3 |
From plate 4: | 4T1 | 4T2 | 4T3 | 4T4 |
W (H2O control) |
PCR Cycles:
- 98C @ 3min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 1201
End: 1305
Gel verification colony PCR products
- Protocol: gel verification protocol in Protocol (SOP)
- Changes: 1% agarose gel
- Machine conditions: 0.5x TBE buffer, 100V, 60min
Gel orientation:
Gel orientation
pT1 | pT2 | 3T1 | 3T2 | 3T3 | 100bp ladder | 4T1 | 4T2 | 4T3 | 4T4 | W (control) |
Results:
Transformation
- Transform 1 and 2 (ligation mixes) from yesterday since there were no colonies on 1 (yesterday) and 2 (4 days ago) --> currently no transformants with His-tag primers
- Protocol: transformation protocol from SOP
- Changes: 10uL of ligation mix instead of 1uL; in 37C incubator for 1.5 hours
- Plated on amp plates and into incubator (37C) @ 1445
ON Culture for miniprep
- Picked colonies 3T1 & 4T3 into 2 tubes
- Put in 37C incubator @ 1606
Vicki Ma 13:56, 11 August 2010 (EDT)
Phage/Phage Standard Track
Eric F.
Prep
- Both O/N culture controls showed no growth
- All bacterial samples showed growth
- Melted Soft+Hard Agar made yesterday
- Soft: 10x40mL Falcon tube aliquots
- Hard: 20x20mL Plates
UV Induction Protocol
- Used SOP finalized yesterday (August 9th) - Strain = 8325-4
- Growth of 1/50 dilution began @ 1108
- No growth control was used in this step due to a lack of flasks aka bad planning
- The 1/50 dilution was 31mL LB Broth w/ 620 microlitres of overnight culture
- Wanted a t=0 reading and 30mL total volume during growth
- t=0 OD600=0.154 using LB Broth as a blank
- Note: Reading may have be done wrong
- Inferring from RN4220 growth curve, OD600=0.6 should be at 1243
- Planned to take reading at 1230
- Took reading at 1240 - OD600 = 1.543
- Began another dilution - hindered by lack of flasks
- Discovered the strain we were working with is 8325-4 NOT 8325
- 8325 has 3 prophages
- 8325-4 has been cured of all 3 prophages
- Experiment was scrapped due to strain mixup
Biofilm Track
Eric F. + Melody Wang
Biofilm Growth Protocol
- Refined the biofilm protocol to a work of art
- Each person (Melody + Eric) began 2 O/N cultures and 1 control
- Overnight cultures were in 3mL of TSB broth in 10mL test tubes
- Strain 1: 8325-4
- Strain 2: RN4220
Plan:
- RN4220 was picked off a TSA plate and will therefore be used to make a biofilm on August 11th
- 8325-4 needs to be spread on a TSA plate before the biofilm growth protocol can start
- Will spread the O/N culture onto a TSA Plate
- All O/N cultures in @ 1745 @ 220 RPM @ 37°C Eric Finlay 12:42, 11 August 2010 (EDT)