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Team:UQ-Australia/Notebook/Project2
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| - | ''' | + | '''20/10/09 - DANJ preparation for sequencing.''' |
| + | |||
| + | :) | ||
| + | |||
| + | |||
| + | '''18/10/09 - DANJ ligation product miniprep''' | ||
| + | |||
| + | Refer to miniprep procedure for protocol. | ||
| + | |||
| + | '''16/10/09 - DANJ ligation product transformation''' | ||
| + | |||
| + | Refer to transformation procedure | ||
| + | |||
| + | ''Results'' | ||
| + | 15 colonies were picked for incubation | ||
| + | |||
| + | |||
| + | '''15/10/09 - In-Gel extraction of PCR products''' | ||
| + | |||
| + | * Only bands corresponding to GroEL and DNAJ were observed. | ||
| + | |||
| + | |||
| + | {| border="1" | ||
| + | |+ Nanodrop Results: | ||
| + | ! Sample name !! Weight !! Concentration (ng/uL) !! 260/280 | ||
| + | |- | ||
| + | ! GRoEL | ||
| + | | 69.5mg || 2.3 || 3.59 | ||
| + | |- | ||
| + | ! DNAJ | ||
| + | | 140.1mg || 2.6 || 2.13 | ||
| + | |} | ||
| + | |||
| + | '''14/10/09 - Gel Electrophoresis for PCR products''' | ||
| + | |||
| + | Lane allocation (30ul samples) | ||
| + | * 1.1kb ladder | ||
| + | * 2. GRoEL | ||
| + | * 3. GRoES | ||
| + | * 4. DNAJ | ||
| + | |||
| + | |||
| + | Results: | ||
| + | * GRoEL = 1658 bp | ||
| + | * GRoES = 371bp | ||
| + | * DNAJ = 1804bp | ||
| + | |||
| + | '''13/10/09 - Digestion of PCR products: GroEL, GroES, DNAJ and Nanodrop''' | ||
| + | |||
| + | * Tube 1: GRoEL + EcoR1 + XhoI | ||
| + | * Tube 2: GRoES + EcoR1 + XhoI | ||
| + | * Tube 3: DNAJ + EcoR1 + Pst1 | ||
| + | |||
| + | |||
| + | {| border="1" | ||
| + | |+ Nanodrop Results: | ||
| + | ! Sample name !! Concentration (ng/uL) !! 260/280 | ||
| + | |- | ||
| + | ! GRoEL | ||
| + | | 410.4 || 1.29 | ||
| + | |- | ||
| + | ! GRoES | ||
| + | |460 || 1.35 | ||
| + | |- | ||
| + | ! DNAJ | ||
| + | | 406.1 || 1.35 | ||
| + | |} | ||
| + | |||
| + | |||
| + | '''09/10/09 - Miniprep of DNAK (mutagenesis FINAL)''' | ||
| + | |||
| + | Procedure: please refer to previous miniprep procedure. | ||
| + | |||
| + | '''07/10/09 - PCR for GroEL mutant, GRoES, and DNAJ''' | ||
| + | |||
| + | AIM: Amplify GRoEL mutant, GRoes and DNAJ plasmids. | ||
| + | |||
| + | '''07/10/09 - Transformation of DNAK mutant into Escherichia coli culture''' | ||
| + | |||
| + | Refer to previous transformation procedure. | ||
| + | |||
| + | '''06/10/09 - P.syringae culture 2''' | ||
| + | |||
| + | 200ml of Nutrient Broth was addded to a conical flask (1000ml) and was innoculated with a loopfull of P.syringae from drystock (from FELIX freezer) | ||
| + | |||
| + | ''Incubation'' | ||
| + | * Agitation = 150RPM | ||
| + | * Temperature = 26 C | ||
| + | * Time = 3 days | ||
| + | |||
| + | |||
| + | '''05/10/09 - Mutagenesis of DNAK (EcoR1 site)''' | ||
| + | Refer to mutagenesis procedure | ||
| + | |||
| + | '''New primers arrived!!''' | ||
| + | * ES forward | ||
| + | * ES reverse | ||
| + | * DNAJ forward | ||
| + | * DNAJ reverse | ||
| + | *DNAK reverse 2 | ||
Latest revision as of 03:27, 22 October 2009
Bioprecipitation Project
20/10/09 - DANJ preparation for sequencing.
- )
18/10/09 - DANJ ligation product miniprep
Refer to miniprep procedure for protocol.
16/10/09 - DANJ ligation product transformation
Refer to transformation procedure
Results 15 colonies were picked for incubation
15/10/09 - In-Gel extraction of PCR products
- Only bands corresponding to GroEL and DNAJ were observed.
| Sample name | Weight | Concentration (ng/uL) | 260/280 |
|---|---|---|---|
| GRoEL | 69.5mg | 2.3 | 3.59 |
| DNAJ | 140.1mg | 2.6 | 2.13 |
14/10/09 - Gel Electrophoresis for PCR products
Lane allocation (30ul samples)
- 1.1kb ladder
- 2. GRoEL
- 3. GRoES
- 4. DNAJ
Results:
- GRoEL = 1658 bp
- GRoES = 371bp
- DNAJ = 1804bp
13/10/09 - Digestion of PCR products: GroEL, GroES, DNAJ and Nanodrop
- Tube 1: GRoEL + EcoR1 + XhoI
- Tube 2: GRoES + EcoR1 + XhoI
- Tube 3: DNAJ + EcoR1 + Pst1
| Sample name | Concentration (ng/uL) | 260/280 |
|---|---|---|
| GRoEL | 410.4 | 1.29 |
| GRoES | 460 | 1.35 |
| DNAJ | 406.1 | 1.35 |
09/10/09 - Miniprep of DNAK (mutagenesis FINAL)
Procedure: please refer to previous miniprep procedure.
07/10/09 - PCR for GroEL mutant, GRoES, and DNAJ
AIM: Amplify GRoEL mutant, GRoes and DNAJ plasmids.
07/10/09 - Transformation of DNAK mutant into Escherichia coli culture
Refer to previous transformation procedure.
06/10/09 - P.syringae culture 2
200ml of Nutrient Broth was addded to a conical flask (1000ml) and was innoculated with a loopfull of P.syringae from drystock (from FELIX freezer)
Incubation
- Agitation = 150RPM
- Temperature = 26 C
- Time = 3 days
05/10/09 - Mutagenesis of DNAK (EcoR1 site)
Refer to mutagenesis procedure
New primers arrived!!
- ES forward
- ES reverse
- DNAJ forward
- DNAJ reverse
- DNAK reverse 2
29/09/09 - 30/09/09 - Overnight digestion of DNAK, GRoEL mutagenesis + Gel electrophoresis
Set up 16 tubes for digestion
- GroEL
- GRoEL + BamH1
- GRoEL + Xba
- GRoEL + BamH1 + Xba
- GRoEL mutant
- DNAK
- DNAK + BGLII
- DNAK mutant 1 + BgLII
- DNAK + EcoR1
- DNAK mutant 1 + EcoR1
- DNAK mutant 2 + BgLII
- DNAK mutant 2 + EcoR1
- DNAK mutant 3 + BgLII
- DNAK mutant 3 + EcoR1
- DNAK mutant 4 + BgLII
- DNAK mutant 4 + EcoR1
Gel Electrophoresis Lane allocation (30ul samples)
- 1kb ladder
- GroEL
- GRoEL + BamH1
- GRoEL + Xba
- GRoEL + BamH1 + Xba
- GRoEL mutant
- DNAK
- DNAK + BGLII
- DNAK mutant 1 + BgLII
- DNAK + EcoR1
- DNAK mutant 1 + EcoR1
- DNAK mutant 2 + BgLII
- DNAK mutant 2 + EcoR1
- DNAK mutant 3 + BgLII
- DNAK mutant 3 + EcoR1
- DNAK mutant 4 + BgLII
- DNAK mutant 4 + EcoR1
29/09/09 - P.syringae culture
A loop of P.syringae cells were added from the dry stock into 50ml of growth media (NYBD)
25/09/09 - Digestion of EL, K(mutant)
Well Allocation (30ul samples)
- cut EL
- cut EL (BamH1)
- cut EL (Xba)
- cut EL (BamH1 + Xba)
- mut EL (BamH1 + Xba)
- 1kb ladder (2ul and 6ul loading dye)
- cut K
- cut K (BglII)
- mut K (BglII)
- cut K (EcoR1)
- mut K (EcoR1)
- mut K (EcoR1)
GEL PICTURE
24/09/09 - Phosphorylation, Mutagenesis, Ligation for K (double mutant)
24/09/09 - DNA Miniprep of EL (mutant)
22/09/09 - Digestion of Miniprep (K - double mutant)
22/09/09 - Colony Pick
One colony was found for the EL transformation. This was picked and incubates at 37'C
21/09/09 - Nutrient Broth, Miniprep and Transformation
It was a busy day,
- Nutrient Broth was made. 13g/L of Nutrient Broth were made (Total of 4L)
- Miniprep (K - double mutant):
- Transformation (K - single mutants):
18/09/09 - Mutagenesis for PXCK-K and PXCK-EL
We have done mutagenesis on two of the plasmids. Here is a quick overview on the process: CLICK HERE
15/09/09 - Transformation with PXCK-K and PXCK-EL
PXCK-K and PXCK-EL plasmids were transformed into electrocompetent E. coli and left overnight at 37C in ampicillin plates (PXCK-K) or chloramphenicol plates (PXCK-EL).
08/09/09 Our Primers have arrived today, this means we can start to put our four genes into the standard plasmids!
02/09/09 For the last couple of weeks we have been trying to design primers in order to put them into the iGEM standard plasmids. Since restriction sites are against us, we are using mutagenesis in order to bypass this problem. Primers were ordered TODAY!
19/08/09 Miniprep of bacterial cultures. For procedure click HERE. Plasmids were stored in -20'C freezer (FIONA).
18/08/09 Colonies were picked from transformed bacteria. Protocol can be found by clicking HERE.
14/08/09
Results from transformation 2
- pxCK-EJ = 1 colony
- pxCK-ES = 3 colonies
- pxCK-EL = 27*4 colonies
- pxCK-K = 53*4 colonies
Plates were sealed with parafilm and stored in the -4 fridge.
13/08/09 A second transformation was performed on the four plasmids. Procedure slightly changed, click HERE to see it.
11/08/09 No results were given from the transformation. Transformation will have to be repeated.
10/08/09
Transformation of our 4 plasmids took place. They were transformed into TOP10 bacteria. A colony check will be preformed tomorrow morning.
To see protocol, click HERE
7/08/09
Vectors were examined and we are planning for the best cloning strategy for the standard plasmids.
6/08/09
Plasmid solutions were left overnight and in the morning checked again with Nanodrop. No DNA was measured. Samples were put in the waterbath at 37 degrees and left for a couple of hours. If Nanodrop does not work after the waterbath we will still do transformation since we only need few plasmids for the transformation to work. Nanodrop may not have been able to quantify such a small concentration of plasmids.
LB media was poured on petri dishes and plasmid DNA was checked again using Nanodrop.
- pXCK-EL = 10.55 ng/uL
- pXCK-ES = 8.43 ng/uL
- pXCK-K = 11.73 ng/uL
- pXCK-E/J = 2.66 ng/uL
Plasmids were stored and transformation will be done on Monday.
5/08/09
Plasmids have arrived!
We have put them into 150 mL of TE buffer and left overnight for the paper to dissolve and later extract the plasmid from the solution.
A Nanodrop was used to see if the DNA was coming out of the paper. No DNA was detected, thus samples were left overnight.
Click on the plasmid name to look at the vector
