IGEM:MIT/2007/Notebook/2007-7-23: Difference between revisions
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#Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids) | #Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids) | ||
#*run overnight | #*run overnight | ||
# | #Dilute sequencing primers | ||
#*sequence PHIE6P | #*sequence PHIE6P | ||
#*sequence F2620+B0034+CPX | #*sequence F2620+B0034+CPX |
Revision as of 12:33, 23 July 2007
Agenda
- Polystyrene Assay
- Morning: get correct OD
- Add AHL
- Afternoon: Test
- Digest I13500
- Ligate 3A with I13500 and Mer in pSB1AT3 (so that, if we combine with polystyrene-binding plasmid into a single cell, can test for presence of both plasmids)
- run overnight
- Dilute sequencing primers
- sequence PHIE6P
- sequence F2620+B0034+CPX
- Order T7 antibody
Digestion of I13500
- Made two mixtures
4.5 µl I13500 (Miniprep #1, 223.5 ng/µl) 1 µl XbaI 1 µl PstI 0.5 µl BSA 5 µl NEB3 Buffer 38 µl H20 ------------------------- 50 µl Total
- Placed in 37C @ 10:20am --> Take out @ 1:20pm
PCR Purification of Digested I13500 (JCH)
- Nanodrop: 23.3 ng/µL
3A Ligation of Mer promoter+Mer regulatory protein (pTO40) and RBS+GFP (I13500) into pSB1AT3 (SK, JH, BH)
Part Length Nanodrop pSB1AT3 3450 bp 125 ng/µl pT040 700 29.9 I13500 740 23.3
- Ligation Mixtures Calculations (see wiki Notebook 7/9)
Backbone: (50 ng) / (125 ng/µl) = 0.4 µl of pSB1AT3 Insert 1: 29.9x = (4)(700/3450)(50) --> x = 1.357 µl of Mer Insert 2: 23.3x = (4)(740/3450)(50) --> x = 1.841 µl of I13500 (cut with X and P)
- Ligation Mixtures
Positive Sample, "iGEM 7/23 Lig + mer+I13500 in 1AT3" 0.4 µl pSB1AT3 1.36 µl pT040 (Mer) 1.84 µl I13500 (RBS+GFP) 0.5 µl ligase (T4) 2.0 µl ligase buffer 10x (T4) 13.9 µl H2O ---------- 20.0 µl total Negative Control (backbone only), "iGEM 7/23 Lig. - in 1AT3" 0.4 µl pSB1AT3 0.5 µl ligase (T4) 2.0 µl ligase buffer 10x 17.1 µl H2O ----------- 20.0 µl total
- Two PCR tubes (+ and -) put in Thermocycler, Block A, OVERNIGHT
- Program name "16IGEM"
- No heated lid
- 16 degrees Celsius, forever
Diluted BioBrick Sequencing Primers
You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room).
Diluted from 100 µM stock to 32.2 µM with total volume of 1.55 mL each.
Made 1 VF2 and 1 VR for iGEM use.
Polystyrene Assay Protocol
- Cell Lines:
- Controls: DH5-alpha (not transformed)
- FHUA PHIE6 --> IPTG
- CPX --> AHL
- Media:
- H20
- PBS
- PBS + Tween + BSA
- Protocol
- Add AHL/IPTG to Liquid Culture
- Wait an hour for AHL induced expression
- Incubate wells in wash media for 30 minutes on rocker at speed 5
- Centrifuge cells out of Liquid Culture media
- Resuspend in wash media
- Add cells to appropriate well
- Place on rocker for 1 hour at speed 5
- Aspirate/Pipet out media in wells