User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/25

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2012/09/25 Entry for User:Sean_P_Corum/Notebook/PHIX174_Cell_Free)
m (Entry title)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.===
-
* Insert content here...
+
-
 
+
* Quantifluore of pBEST-Pr-MG-apt-UTR1-deGFP-T500 =  nM.
-
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
+
* Do cell-free expression in batch-mode to characterize pBEST-Pr-MG-apt-UTR1-deGFP-T500. Idea is to start at lowish gain (say 50), and then increase gain at ~3-4 hr when expression is peaking until an 80% of max reading (80,000 counts of 100,000 max for the PMT) is achieved. This will then be the fixed gain for MGapt experiments.
-
|}
+
* Cell-free expression 80%:
-
 
+
** 30 μL extract (12May11)
-
__NOTOC__
+
** 12.1 μL water
 +
** 0.9 μL 100 mM Mg-glutamate (1 mM final)
 +
** 2 μL 3 M K-glutamate (66.7 mM final)
 +
** 22.5 μL 6 mM amino acids mix (1.5 mM final)
 +
** 4.5 μL PEG8000 40% (2% final)
 +
* Aliquot 6 × 9 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
 +
*# 1 μL water = 0 nM
 +
*# 1 μL 50 nM / 3^4 * 20% = .123 nM
 +
*# 1 μL 50 nM / 3^3 * 20% = .370 nM
 +
*# 1 μL 50 nM / 3^2 * 20% = 1.11 nM
 +
*# 1 μL 50 nM / 3^1 * 20% = 3.33 nM
 +
*# 1 μL 50 nM / 3^0 * 20% = 10 nM
 +
* Plate reader:

Revision as of 13:10, 25 September 2012

PHIX174 Cell Free Expression Main project page
Previous entry      Next entry

Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Quantifluore of pBEST-Pr-MG-apt-UTR1-deGFP-T500 = nM.
  • Do cell-free expression in batch-mode to characterize pBEST-Pr-MG-apt-UTR1-deGFP-T500. Idea is to start at lowish gain (say 50), and then increase gain at ~3-4 hr when expression is peaking until an 80% of max reading (80,000 counts of 100,000 max for the PMT) is achieved. This will then be the fixed gain for MGapt experiments.
  • Cell-free expression 80%:
    • 30 μL extract (12May11)
    • 12.1 μL water
    • 0.9 μL 100 mM Mg-glutamate (1 mM final)
    • 2 μL 3 M K-glutamate (66.7 mM final)
    • 22.5 μL 6 mM amino acids mix (1.5 mM final)
    • 4.5 μL PEG8000 40% (2% final)
  • Aliquot 6 × 9 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
    1. 1 μL water = 0 nM
    2. 1 μL 50 nM / 3^4 * 20% = .123 nM
    3. 1 μL 50 nM / 3^3 * 20% = .370 nM
    4. 1 μL 50 nM / 3^2 * 20% = 1.11 nM
    5. 1 μL 50 nM / 3^1 * 20% = 3.33 nM
    6. 1 μL 50 nM / 3^0 * 20% = 10 nM
  • Plate reader:
Personal tools