Team:UQ-Australia/Notebook/Project2



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=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.

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

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!
 * pXCK-E/J
 * pXCK-K
 * pXCK-EL
 * pXCK-ES

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