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<p>Samples from prior colony PCR were analysed with a 0.8% Agar gel electrophoresis. </p>
 
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Revision as of 16:48, 13 October 2018


Receptor Assay

August
September

BRET Assay

August
September

Methylation Assay

July
August
September
Mo
Tu
We
Th
Fr
Sa
Su

Morning

Performed Calibration 1, 2, and 3 according to protocol. All measurements were performed using the plate reader of Seino.

Table 1. content 96 wells plate cal.1 and cal.2
123456789101112
ALdd
BLdd
CLdd
DLdd
E MSdddddddddddd dddddddddd
FMSdddddddddddd dddddddddd
G MSdddddddddddddddddddddd
H MSdddddddddddddddddddddd
Calibration 1
  • L= 100 uL Ludox CL-X (stored at 4C)
  • dd= 100 uL ddH20
  • Measurement: Abs600, turn off pathlength correction
Calibration 2
  • MS= 200 ul Microsphere Stock Solution
  • dd= 100 uL ddH20
  • green= serial dilution was performed with a micropipet from E1,F1,G1,H1 - E11,F11,G11,H11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
  • Measurement: Abs600, re-mix befor putting in plate reader and prevent bubbles, path length correction off
Calibration 3
  • 1xFC= 200 mL 1xFC (100uL 10x fluorescein + 900ul 1x PBS pH 7.4, tube was covered with foil
  • P= 100 uL 1x PBS pH 7.4
  • green= serial dilution was performed with a micropipet from A1,B1,C1,D1 - A11,B11,C11,D11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
  • Measurement: FL, 530nm/30nm bandpass, 25-30nm with recommened excitation of 485nm, emission 520-530nm of the filter. Path length correction was turned off
Table 2. content 96 wells plate cal.3
123456789101112
A1xFCPPPPPPPPPPP
B1xFCPPPPPPPPPPP
C1xFCPPPPPPPPPPP
D1xFCPPPPPPPPPPP
E
F
G
H

Afternoon

LBC plates were made according to the protocol used on the wall
  • 250ml LB 2x added to melted 250 ml WA 2x using a microwave
  • 0.5ml was added to final solution
  • plates were dried in 37C incubator
Transformation device 3 + negative control interlab study
  • Device 3 (number 5) showed a low GFP expression, so it was tried to re-preform the tranformation. Negative control of the interlab (number 1) was not performed last time due to lack of LBC plates so was also performed.
  • Protocol Transformation
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21/08/18

The Cu-promotor (pSB1K3) and RFP (pSB1K3) were transformed from the kit according to the protocol. The bacteria were plated in 100% and 500%. The custom receptor was ordered and put in the pBSK vector

22/08/18

Bacteria grown and colonies were picked and inoculated in 5 ml LB with

  • Cu-promotor (pSB1K3) = Kanamycin
  • RFP (pSB1K3) = Kanamycin
  • Custom Receptor pBSK = Ampicillin

23/08/18

Miniprep was performed according to the protocol

  • Cu-promotor → 250
  • Custom receptor → 360
  • RFP → 91.7

The “dirty” method of cloning was used to couple the Cu-Promotor and RFP in one vector. The custom receptor was transformed to another vector (pSBK)

For each of the three samples a mastermix was made. For each digestion the DNA concentration was reduced to 25 ng/ul. 4 ul of sample was transferred to a PCR tube with 4 ul of the according restriction mix. The samples were incubated for 1 hour at 37 C. After 1 hour the samples were taken out of the incubator and enzymes were heat inactivated at 80C for 20 min. 1ul of Cu-promotor and 1ul of RFP were added to one PCR tube. Furthermore 1ul DNA T4 ligase buffer and 0.5 ul of T4 ligase were added. The total volume was made up to 10 ul with 3.5 ul dH2O. The samples were incubated for one hour (RT). Enzymes were deactivated with heat kill 80C 20 min.

DNA was transformed in DH5a according to the NEB protocol.

24/08/18

Transformation was successful. Colonies were picked for colony PCR to identify whether the right construct was transformed. 8 colonies of each plate were selected (Cu-promotor + RFP 100%, Cu-promotor + RFP 500%, Custom receptor 100, Custom Receptor 500). Each colony was incubated in PCR tubes with 50 ul LB without antibiotic for 1 hour.

27/08/18

Samples from prior colony PCR were analysed with a 0.8% Agar gel electrophoresis.

1-8 Custom receptor 100%
9-16 Custom receptor 500%
17-24 Cu + RFP 100%
25-32 Cu + RFP 500%

28/08/18

The results obtained in the gel electrophoresis DNA analysis (27/8/18) was not satisfactory. Therefore four new colonies were picked for colony PCR and put in 50 µl LB and incubated for 1 hour from each of the following plates: Cu + RFP 100%, Cu + RFP 500%, Custom Receptor 100%, Custom Receptor 500%. 2 µl was of each pick was transferred to new PCR tubes with the 18 µl of following mix:

Content Volume (µl)
Pol TAQ 0.1
buffer 4
dNTP 0.4
Forward primer 0.4
Revere primer 0.4
H2O 12.7

29/08/18

The DNA of the (28/08/18) samples was purified according to the miniprep protocol.

Sample Concentration ng/µl Description
jan-00 69.9 Cu-Promotor+ RFP
9 77.5 Custom receptor
12 91.8 Custom Receptor
13 49.4 Custom Receptor
14 85.4 Custom Receptor

The prior prepared clonation (28/08/18) was transformed in DH5α according to the iGEM protocol.

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lskdfjslkjgdlkdfjgldkjlakmflsvmclkxmfklmgdmlkcvmblcmkb
ksjdfljslfkdjlkghjfglhkjflhgkjflhgjflghjflghkjflhj
lskdfjslkjgdlkdfjgldkjlakmflsvmclkxmfklmgdmlkcvmblcmkb