Revision as of 14:52, 16 October 2010

Lab notes (9/27 - 10/3)

Flagella Group

Colony PCR of FlhDCmut and B0015 inserted into pSB3T5 with J13002

Date: 9/27 2010
Done By: Sheila
Protocol:
Notes: 34 colonies were tested. VF2 and VR primers were used.
Results:
Conclusion: Another round of colony PCR will be performed tomorrow. If this proves unsuccessful a new ligation and transformation will be performed.

Second colony PCR of FlhDCmut and B0015 inserted into pSB3T5 with J13002

Date: 9/28
Done By: Louise
Protocol:
Notes:
Results:
Conclusion:

Insert clever content here.

Retinal

Coloni PCR on transformations of NinaB in PSB1C3, K343005 in PSB3C5 and K343005 in PSB1AK3 with the dobbelt terminator

Date: 30/09
Done by: Tommy
Methods: PCR
Protocols: CP1.3[1]
Notes:
Because the first coloni PCR of the transformants didn't give us the the right pieces 32 new colonies where chosen:
32-40: ninaB in PSB1C3
41-48: K343005 in PSB3C3
49-64: k343005 in PSB1AK3

VF2 and VR used as primers in the TAQ PCR:

PCR Program:

Start	94 C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	2:30 min
Goto2	rep	29x
End	72 C	5 min
Hold	4 C

Colonies 34, 37, 61 and 63 was selected for minipreps and freezing cultures.

dobbelt transformation of K343005 in PSB1C3 and K274210 in PSB1A2 in to top10 and MG1655 E. Coli

Date: 1/10
Done by: Tommy & Marie
Methods: transformation
Protocols: TR1.1[2]
Notes:
Top10 and MG1655 E. coli competent cells where prepared according to protocol
Minipreps of K343005 and K274210 was used
1uL of K343005 and 3uL of K274210 was used becaus og their differens in concentration. The rest was done accordin to the transformations protocol.

Coloni PCR on dobbelt transformants of K343005 in PSB1C3 and K274210 in top10 and MG1655 E. coli

Date: 2/10
Done by: Tommy
Methods: Coloni PCR
Protocols: CP1.3[3]
Notes:
8 Colonies from each strain of E. Coli where chosen:
1-8: K343005 in PSB1C3 and K274210 in PSB1A2 in top10
9-16: K343005 in PSB1C3 and K274210 in PSB1A2 in MG1655
VF2 and VR used as primers in the TAQ PCR:

PCR Program:

Start	94 C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	5 min
Goto2	rep	29x
End	72 C	5 min
Hold	4 C

Colonies 2, 7, 11 and 12 was selected for minipreps and freezing cultures.

Miniprep on ninaB in PSB1C3 and K343005 in PSB1AK3

Date: 2/10
Done by: Tommy
Methods: Miniprep
Protocols: MP1.2[4]
Notes:
Colonies 34 and 37 contain ninaB in PSB1C3
Colonies 61 and 63 contain K343005 in PSB1AK3
The miniprep was done according to protocol
The minipreps was dryed down and sendt to seqvensing

Further Characterization of BBa_K274210 in MG1655 and TOP10

Date: 3/10
Done by: Tommy
Methods: Miniprep
Protocols: MP1.2[5]
Notes:
Further characterization of the BioBrick K274210 has been performed in Top10 and MG1655 E. coli. The experimental data have shown somewhat similar results for Top10 and MG1655 E. coli. The experiment was performed as follows:
Over night (ON) cultures were grown from 4 colonies, until the following OD’s were obtained:

Top 10 - no insert OD = 0,008 (100 x diluted)
Top 10 - with K274210 insert OD = 0,011 (100 x diluted)
MG1655 - no insert OD = 0,020 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)

ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin. All ON cultures were grown for 20 hours at 37 °C. After 16 hours, 10 ml of the ON cultures were transferred into 110 ml LB media and grown for 4 hours to reach the exponential phase, where the following OD’s were obtained:

Top 10 - no insert OD = 0,049 (100 x diluted)
Top 10 - with K274210 insert OD = 0,044 (100 x diluted)
MG1655 - no insert OD = 0,007 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)

Cultures with K274210 insert were grown in media containing ampicillin. 100 mL cell culture were centrifuged for 5 min at 14000 RPM. The supernatant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the Top 10 E. coli with the K274210 insert, which was resuspended in 10 mL acetone (source of error). The resuspended cells were sonicated for 5 min. Samples were spun down, the supernatant was transferred to new tubes, and cell debris was discarded. A standard curve was made from pure beta-carotene.

The samples at the OD’s seen above as well as solutions of pure beta-carotene with known concentrations were measured at a fixed wavelength of 456 nm. Known concentrations and their absorbances:

Concentration	Absorbance
1 mM	4,000
100 µM	2,260
50 µM	4,000
25 µM	0,155
10 µM	0,893
5 µM	0,440
1 µM	0,075
100 nM	0,015
10 nM	0,038
1 nM	0,005
100 pM	0,024

The samples and their absorbances:

	Top 10 cells (Absorbance)	MG1655 E. coli mutant (Absorbance)
Stationary phase control	0,034	0,024
Stationary phase with K274210 biobrick insert	0,319	1,549
Expotential phase control	0,020	0,024
Expotential phase with K274210 biobrick insert	0,034	0,033

UV-VIS absorbance spectra of the known solutions were obtained, as well as spectra of the samples at the ODs shown above. The spectra of Top 10 and MG1655 in the stationary phase as well as selected spectra of known solutions are shown below:

These results were obtained from one experiment, which will be replicated later for more precision.

Team:SDU-Denmark/labnotes12

From 2010.igem.org