Team:Goettingen/NoteBook 16
From 2013.igem.org
Platereader assay of RiboA with c-di-AMP and Polyamine
Platereader assay of RiboA with c-di-AMP and Polyamine
RiboA 5.8.I and CFP CI in BL-21 and a BL-21 wt were inoculated in an ON culture. At 10am a synchronization culture for each was set up with an StartOD of 0.5.
At 11:30am each well of the 96 well plate was inoculated with a start OD of 0.05.
Setup:
RiboA no c-di-AMP no PA |
RiboA 10nmol c-di-AMP no PA |
RiboA 100nmol c-di-AMP no PA |
RiboA 1000nmol c-di-AMP no PA |
RiboA 10mmol c-di-AMP no PA |
RiboA no c-di-AMP + PA |
RiboA 10nmol c-di-AMP + PA |
RiboA 100nmol c-di-AMP + PA |
RiboA 1mmol c-di-AMP + PA |
RiboA 10mmol c-di-AMP + PA |
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CFP no c-di-AMP no PA |
CFP 10nmol c-di-AMP no PA |
CFP 100nmol c-di-AMP no PA |
CFP 1mmol c-di-AMP no PA |
CFP 10mmol c-di-AMP no PA |
CFP no c-di-AMP + PA |
CFP 10nmol c-di-AMP + PA |
CFP 100nmol c-di-AMP + PA |
CFP 1mmol c-di-AMP + PA |
CFP 10mmol c-di-AMP + PA |
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wt |
wt |
wt |
LB BLK |
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Wt + PA |
Wt + PA |
Wt + PA |
LB BLK+PA |
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Application of some qRTPCR probes onto a gel
Application of some qRTPCR probes onto a gel
in order to see weather different melting temperatures in the qRTPCR make a difference
following probes were used:
83°C: B3, C3, E5
83,5°C: B5, D5, E7
Gel doc:
wells: B3, C3, E5, B5, D5, E7
Repeat of Platereader with DarR + c-di-AMP
Same setup as on the 12.9. Started at 12 o'clock
Click to view full resolution
Readied parts for shipment
Ribo A-D, Ribo A 5.8.1, A1, 6.1 and P7 minipreps were diluted to a concentration of 25ng/µl and a volume of 10 µl.
Stored in a Eppi-rack at -20°C in the top shelf of the freezer. To be marked for shipment
Summary:
1.qRT-PCR together with Katrin G. according to the method collection protocol
2. Competition/Feeding experiment
qRT-PCR together with Katrin G. according to the method collection protocol
10 µl 2X SYBR Green reaction mix (light sensitive)
0.4 µl Reverse Transcriptase
x µl RNA (100ng/20 µl)
x µl H2O
Total 17.6 µl
- Final reaction (total volume: 20 µl) should contain 100 ng RNA, 10 µl 2x buffer, 0.4 µl enzyme and 1.2 µl of each primer (1:20 diluted in RNase free water)
- Mastermixes for each RNA sample and a neg. control were prepared. They consisted of2x buffer (light-sensitive – contains SYBRgreen!), RNA/RNase free water for no-template-controls, RNase free water, and enzyme for 3.5 reactions.
- Mastermixes consisting of 1.2 µl Primer fwd and 1.2 µl Primer reverse per reaction were prepared and then added to 2.4 µl primer mix prepared in the wells of the qRT-PCR plate
- 17.6 µl of the buffer/template/water/enzyme mastermix were added to the wells
- qRT-PCR run using standard protocol
RFP primers (iGEM_98 and iGEM_99) and rpoA primers (iGEM_102 and iGEM_103) were used
Calculations:
Pipetting sceme:
Results:
Competition/Feeding experiment
OD measurements of ON culture
Ribo A 5.8.1 |
CFP control |
DAC |
DAC CTRL |
2,34 |
4,2 |
6,17 |
4,33 |
All cultures were synchronised to OD 0.5. After 2,5 h, fresh media was inocculated with a single strain or mixed. For each, one doublicate with IPTG was set up.
Cultures were inocculated to an OD of 0.1. For the mixed cultures, each culture was added to an OD of 0.05. After 3h those ODs were meassured.
Culture |
DAC CTRL |
DAC |
Ribo A |
CFP |
CFP/DAC |
CFP/CTRL |
RiboA/DAC |
RiboA/CTRL |
OD -IPTG |
1,663 |
1,68 |
1,343 |
1,644 |
1,685 |
1,63 |
1,603 |
1,576 |
OD + IPTG |
0,532 |
1,574 |
1,371 |
1,639 |
1,627 |
1,507 |
1,530 |
1,603 |
From these cultures, a dilution series of 1 cell/µl was set up.
These dilutions were plated onto +/- IPTG + XGal LB plates with 50 cells per half. On one half of the according plate, the premixed cultures were streaked. On the other half, the same mixes were plated from the isolated grown cultures with 25µl (25 cells) per culture. Every single culture was plated seperate as well.
Plates were cultivated at 37°C in the dark over night.
Pictures taken next day
Click here to see the result
Summary:
1. DNAse digest, Control PCR
2.Inocculation of ON cultures
DNAse digest
2500 ng RNA |
respectively |
DNAse |
2,5 µl |
10x buffer |
2,5 µl |
water |
rest |
Total |
25 µl |
stored in heat block 30 min at 37°C
then EDTA was added 2,5 µl per probe and put into thermo cycler at 65°C for 10 min
Control PCR
- final reaction composition:
Component |
Volume |
Template (RNA or gDNA) |
1 µl |
Primer fwd 1:20 iGEM 102 |
1 µl |
Primer rev 1:20 iGEM 103 |
1 µl |
PfuS |
0.5µl |
dNTPs |
1 µl |
5x HF buffer |
5 µl |
water |
15,5µl |
total |
25 µl |
in thermo cycler protocol: Ivonne -> PhuS
Gel doc:
wells: Part1C3exp, part2C3exp, part3C3exp, part4C3exp, part8C3exp, Part1C3stat, part2C3stat, part3C3stat, part4C3stat, part8C3stat; Part1C3ON, part2C3ON, part3C3ON, part4C3ON, part8C3ON, gDNA
Nanodrop:
Inocculation of ON cultures
Ribo A 5.8.1, CFP religant, DAC and DAC control inocculated 4ml each for feeding/competition experiment