IGEM:IMPERIAL/2007/Experimental Design/Phase2/Protocol 1.2: Difference between revisions

Revision as of 03:05, 3 September 2007

Status:

Aims:

Equipment Reagents

Protocol

Be careful about exposing the GFP samples to light to prevent photo bleaching and in addition kept on ice to prevent degradation
Remove the sample of purified GFP from the feezer and place in a container at room temp, to allow to melt
Remove 10ul of purified GFP and add to 27ul of dii₂0 in an PCR tube. This gives a 37ul of 10uM GFP concentration.
Remove 2ul of 10uM GFP to 18ul of dii₂0 in an PCR tube.This gives a 10ul of 1uM GFP concentration
Remove 8ul of 1uM GFP to 8ul of dii₂0 in an PCR tube.This gives a 16ul of 0.5uM GFP concentration
Remove 8ul of 0.5uM GFP to 8ul of dii₂0 in an PCR tube.This gives a 16ul of 0.0.25uM GFP concentration
Remove 8ul of 0.25uM GFP to 8ul of dii₂0 in an PCR tube.This gives a 16ul of 0.125uM GFP concentration
Keep all samples on ice and in darkness
Repare the cell extracts

Reagents

Our Prepared S30 extract
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
Commercial S30 T7 extract. Including:
- 175µl Amino Acid Mixture Minus Cysteine, 1mM
- 175µl Amino Acid Mixture Minus Methionine, 1mM
- 175µl Amino Acid Mixture Minus Leucine, 1mM
- 450µl T7 S30 Extract, Circular (3 × 150µl)
- 750µl S30 Premix Without Amino Acids
MiiA water x1ml
GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)
Prepared dilutions of AHL to 1mM

Reagents

Recombinant GFP (Mw=27,000Da) Link to product information. It is in the following form;
100ug in buffer of 1mg/ml, this means in the solution there is:
- 3.7x10^-9 moles of GFP
- 0.037 M (moles dm^-3)
- Volume supplied to us is 0.1mL

Protocols

The moles of GFP that our in vitro systems is expected to produce is 7.4x10^-12 moles, this is based upon the quoted expected protein production of 200ng (2x10^-7/27000Da). If the total volume of the cell extract is 100ul, this means that the concentration is around 7.4x10^-8 M.
We need a sufficient range of dilutions to cover this range. However, the relationship between fluorescence and molcules of GFP is linear at concentrations in uM and lower. Therefore, if we have a sufficient range we should be able to plot an accurate calibration curve.
We propose covering the following range of 8 data sets with 2 repeats for each:

1.000uM
0.500uM
0.250uM
0.125uM
0.060uM

@@ Line 6: / Line 6: @@
 *To create and test a series of dilutions of GFP using a purified sample of GFP
 '''Equipment'''
+'''Reagents'''
+*Pure sample of recombinant GFP
+*DiiH<sub>2</sub>0
+*PCR tubes
+'''Protocol'''
+#Be careful about exposing the GFP samples to light to prevent photo bleaching and in addition kept on ice to prevent degradation
+#Remove the sample of purified GFP from the feezer and place in a container at room temp, to allow to melt
+#Remove 10ul of purified GFP and add to 27ul of dii<sub>2</sub>0 in an PCR tube. This gives a 37ul of 10uM GFP concentration.
+#Remove 2ul of 10uM GFP to 18ul of dii<sub>2</sub>0 in an PCR tube.This gives a 10ul of 1uM GFP concentration
+#Remove 8ul of 1uM GFP to 8ul of dii<sub>2</sub>0 in an PCR tube.This gives a 16ul of 0.5uM GFP concentration
+#Remove 8ul of 0.5uM GFP to 8ul of dii<sub>2</sub>0 in an PCR tube.This gives a 16ul of 0.0.25uM GFP concentration
+#Remove 8ul of 0.25uM GFP to 8ul of dii<sub>2</sub>0 in an PCR tube.This gives a 16ul of 0.125uM GFP concentration
+#Keep all samples on ice and in darkness<br><br>
+#Repare the cell extracts
 *Fluorometer + Connected PC Turn on before beginning
 *96 well plate x1 + Plate lid