Imperial/Wet Lab/Protocols/ID1.1

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= Wet Lab: Protocols: Initial Testing for DNA Constructs ''in vivo'' =

'''Aims''' 
*To test to see if the DNA construct from the registry are viable. This is done in vivo.
*The constructs are pTet-LuxR-pLux-GFP.
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===Day 1===
====Equipment====
*7ml sterile tubes x4
*1.5ml Eppendorf tube x1
*37&deg;C incubator
*Gilson Pipettes p1000 p200 p20

====Reagents====
*''E.coli'' BL21; culture containing pTet-LuxR-pLux-GFP
*LB medium

*Ampicillin stock (50 mg/ml)
*AHL stock
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'''Innoculation of Media'''
#Inoculate 10ul of stored parts in individual 2ml LB medium containing 2ul of ampicillin
#Incubate at 37°C for overnight in a shaker. '''(This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase)'''
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'''Preparation of culture for AHL induction and GFP measurement'''
From the stock culture of transformed E.coli, dilute the culture so that the OD=0.1.


'''Preparation of diluted series of AHL'''<br>
http://partsregistry.org/wiki/images/4/4c/F2620-TF-Small.png <br>
'''(Taken off [http://partsregistry.org/Part:BBa_F2620 BBa_F2620]. Results show required dilution of AHL solution)'''

#Add 6µl of AHL stock solution to 600µl of cell samples, to a final concentration of 1uM. 
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===Day 2===
====Equipment====
*37&deg;C incubator
*Fluorometer + PC
*Gilson pipettes 1000 and 200
*1 Fluorometer plate (black)
*Eppendorf Tubes
*Stop watch

====Reagents====
*LB medium
*E.coli culture with transformed plasmid
*10μM AHL stock
*100μM AHL stock
*GFP stock solution
*ddH2O
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====Protocol====
'''Preparation of diluted GFP standard solution'''<br>
#Add 995ul of ultra pure water an eppendorf tube, together with 5ul of undiluted GFP standard solution and mix. '''(This gives a 200x dilution to be used as a positive control)''' 
#Place the tube on ice till it is ready to be used.
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'''Loading Plate'''
1. Transfer 200 µl aliquots of each of the cultures to a flat-bottomed 96 well plate. '''(Follow the schematic as shown)'''
: Three wells to be filled with 200µl of media to measure the absorbance background.
: Three further wells to be filled with 200 µl of (media + empty-vector culture) to measure fluorescent background. 
: Standard GFP solution added as a positive control.

2. Add Xµl of  stock concentration of AHL to the respective wells as shown.
3. Incubate it at 37oC for 4 hours.
4. Remove lid and measure in the flourometer. 
: (Fluorescence measurements - 488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units)
5. Repeat the measurement a further two times straight after each other '''(This is to test the variability of the machine)'''
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'''Schematic'''
{| border="1" cellpadding="1"
|
{| border="1" cellpadding="2"
!<u>Well </u> || <u>Test Construct</u> || <u>Stock Volume (ul)</u> !! <u>AHL (ul)</u> !! <u>Final [AHL]</u>
|-
|<font color="blue">A1 
|<font color="blue">pTet-LuxR-pLux-GFP + 1μM AHL
|<font color="blue">200
|2
|10<sup>-6</sup>M
|-
|<font color="blue">A2
|<font color="blue">pTet-LuxR-pLux-GFP + 1μM AHL
|<font color="blue">200
|2
|10<sup>-6</sup>M
|-
|<font color="blue">A3
|<font color="blue">pTet-LuxR-pLux-GFP + 1μM AHL
|<font color="blue">200
|2
|10<sup>-6</sup>M
|-
|<font color="blue">A5
|<font color="blue">LB-Amp Media
|<font color="blue">200
|0
|0
|-
|<font color="blue">A6
|<font color="blue">LB-Amp Media
|<font color="blue">200
|0
|0
|-
|<font color="blue">A7
|<font color="blue">LB-Amp Media
|<font color="blue">200
|0
|0
|-
|<font color="blue">B1
|<font color="blue">pTet-LuxR-pLux-GFP
|<font color="blue">200
|0
|0
|-
|<font color="blue">B2
|<font color="blue">pTet-LuxR-pLux-GFP
|<font color="blue">200
|0
|0
|-
|<font color="blue">B3
|<font color="blue">pTet-LuxR-pLux-GFP 
|<font color="blue">200
|0
|0
|-
|<font color="blue">B5
|<font color="blue">LB-Amp Media + Non-expressing culture
|<font color="blue">200
|0
|0
|-
|<font color="blue">B6
|<font color="blue">LB-Amp Media + Non-expressing culture
|<font color="blue">200
|0
|0
|-
|<font color="blue">B7
|<font color="blue">LB-Amp Media + Non-expressing culture
|<font color="blue">200
|0
|0
|-
|<font color="blue">C5
|<font color="blue">Diluted GFP Solution
|<font color="blue">200
|0
|0
|-
|<font color="blue">C6
|<font color="blue">Diluted GFP Solution
|<font color="blue">200
|0
|0
|-
|<font color="blue">C7
|<font color="blue">Diluted GFP Solution
|<font color="blue">200
|2
|10<sup>-6</sup>M
|}
|
[[Image:Working7.png|450px|top|In vivo Testing 96 well plate]]
|}
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