Imperial/Wet Lab/Protocols/CBD1.3

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=Protocols for DNA concentration experiments=
Experiments to be carried out are to determine the optimum concentration of the CBD construct, in-vitro, so that we get the highest level of protein expression after a period of 6hours. The construct to be tested is pTet-GFP.  <br>
<br>The concentrations of DNA that will be tested are: 1, 2, 4 and 6&micro;g. Samples will be kept at 37°C.

==Aims==
*To determine the concentration of pTet construct for which output is optimum, in terms of the reponse time and the output fluorescence at the end of the experiment time.

==Equipment==
*Fluorometer + PC 
*37°C incubator 
*Fluorometer plate  (black)
*Sticky seal tape
*Gilson pipettes 200, 20, 10 
*Eppendorf Tubes x 7
*Stopwatch
*Foil

==Reagents==
*Commercial S30 E.coli extract. Including:
**175µl Amino Acid Mixture Minus Cysteine, 1mM
**175µl Amino Acid Mixture Minus Methionine, 1mM
**175µl Amino Acid Mixture Minus Leucine, 1mM
**450µl S30 Extract, Circular (3 × 150µl)
**750µl S30 Premix Without Amino Acids
*Nuclease Free water x1ml
*DNA pTet-GFP from midiprep

==Preparation of reactions==
#First collect all equipment and reagents and ensure that the fluorometer and the PC connected has a data collection protocol installed. 
#Place the 96-well plates into the 37&deg;C incubator.
#For the cell extract, get the following out of the cell extract kit:
#*A.A's from kits 
#*Premix tube
#*S30 tubes
#To prepare the commercial E.coli Cell Extract, carry out the following procedure, two times:<br>
##First prepare a complete amino acid mixture for the extract solution: Add the 25µl volume of two amino acid minus mixtures into an labeled eppendorf to give a volume of 50µl. Each amino acid minus mixture is missing one type of amino acid. 
##Take an eppendorf tube and add the 50µl of the E.coli complete amino acid mixture. 
##Add 200µl of S30 Premix (Without Amino Acid) into the eppendorf tube. 
##Then add 150µl of S30 Extract Circular too. 
##The final volume of cell extract is: 400&micro;l 
#Each cell extract will be used to test one of the constructs. Label the tubes, identifying which construct it will be used for.
#Incubate cell extract mixture for CBD in the 37&deg;C incubator.
#Prepare the different DNA concentrations for pTet construct (concentration of pTet DNA = 500ng/&micro;l): 
##Concentration 1 = 1&micro;g: Add 4&micro;l of DNA in 36&micro;l nuclease free water.
##Concentration 2 = 2&micro;g: Add 8&micro;l of DNA in 32&micro;l nuclease free water.
##Concentration 3 = 4&micro;g: Add 12&micro;l of DNA in 28&micro;l nuclease free water.
##Concentration 4 = 6&micro;g: Add 16&micro;l of DNA in 16&micro;l nuclease free water.
#This will give a total volume of 40µl of each DNA concentration. Put each DNA into a seperate, labeled eppendorf tube and place them in the 37&deg;C incubator.

===Loading Plate===
#Take the plate out of the incubation.
##Follow the schematic for the plate 1 (37&deg;C incubator) and begin by loading 40&micro;l of the in vitro expression system into the right wells. 
##Tap down the top of the plate to bring down any solution to bottom of the well. 
##Then add 20µl of purified DNA sample to each well, as indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
##Add 20&micro;l of nuclease free water into the two negative control wells, as shown in the schematics.
#Put 60&micro;l of water into some empty wells in the middle of each plate. These will be used to check for evaporation. 
#After the DNA and the cell extract mixtures have been put into their respective wells, load the program on the PC to measure the fluorescence in the right wells. 
#Create a file with name referring to the temperature of the plate, under: D:\IGEM\'''INSERT DATE'''\CBD\ OTR , for CBD construct. The data from the fluoreometer will be exported here. 
#Each file with the reading should be named as the following: 
#*construct-temp-time-date 
#Place the plate in the fluorometer to measure its initial fluorescence reading. 
#After the measurement, place the sticky tape across the plate, and put the plate in the 25oC water bath.
#Start on the next plate, and repeat procedures 2-6.
#Place the plate in the 37oC incubator.
#Before placing them in the incubator, wrap aluminium foil around them to prevent photobleaching. 
#Measure the temperature every 30 minutes for each temperature, for 6 hours.

==Schematic==

===Plate 1===

{| border="1" cellpadding="1"
!<u>Well</u> || <u>Test Construct</u> !! <u> Concentration of DNA</u> !! <u>In vitro chassis</u> 
|-
|<font color=blue> E5
|<font color=blue> Nuclease Free Water (Negative control)
|<font color=blue> 0&micro;g 
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> E7
|<font color=blue> Nuclease Free Water (Negative control)
|<font color=blue> 0&micro;g 
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> C3
|<font color=blue> pTet-GFP
|<font color=blue> 1&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> C5
|<font color=blue> pTet-GFP
|<font color=blue> 1&micro;g
|<font color=blue>Commercial E.coli extract 
|-
|<font color=blue> C7
|<font color=blue> pTet-GFP (positive control)
|<font color=blue> 2&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> C9
|<font color=blue> pTet-GFP (positive control)
|<font color=blue> 2&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> D4
|<font color=blue> pTet-GFP
|<font color=blue> 4&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> D6
|<font color=blue> pTet-GFP
|<font color=blue> 4&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> D8
|<font color=blue> pTet-GFP
|<font color=blue> 6&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|<font color=blue> D10
|<font color=blue> pTet-GFP
|<font color=blue> 6&micro;g
|<font color=blue> Commercial E.coli extract 
|-
|}

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