IGEM:MIT/2007/Notebook/2007-7-23: Difference between revisions
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#Digest I13500 | #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) | #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 | #Order T7 antibody | ||
Line 17: | Line 17: | ||
<pre> | <pre> | ||
4. | 4.5 µl I13500 (Miniprep #1, 223.5 ng/µl) | ||
1 µl XbaI | |||
1 µl PstI | |||
0. | 0.5 µl BSA | ||
5 µl NEB3 Buffer | |||
38 µl H20 | |||
------------------------- | ------------------------- | ||
50 µl Total | |||
</pre> | </pre> | ||
* Placed in 37C @ 10:20am --> Take out @ 1:20pm | * 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)== | |||
<pre> | |||
Part Length Nanodrop | |||
pSB1AT3 3450 bp 125 ng/µl | |||
pT040 700 29.9 | |||
I13500 740 23.3 | |||
</pre> | |||
*Ligation Mixtures Calculations (see wiki Notebook 7/9) | |||
<pre> | |||
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) | |||
</pre> | |||
*Ligation Mixtures | |||
<pre> | |||
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 | |||
</pre> | |||
*Two PCR tubes (+ and -) put in Thermocycler, Block A, OVERNIGHT -- started at 3 PM | |||
**Program name "16IGEM" | |||
**No heated lid | |||
**16 degrees Celsius, forever | |||
==Diluted BioBrick Sequencing Primers== | ==Diluted BioBrick Sequencing Primers== | ||
You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room). | You can find the stock eppendorfs in the unnamed -20C fridge (Jason+Barry's room). | ||
Diluted from | 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. | Made 1 VF2 and 1 VR for iGEM use. | ||
Line 37: | Line 79: | ||
==Polystyrene Assay Protocol== | ==Polystyrene Assay Protocol== | ||
* Controls: DH5-alpha (not transformed) | * Cell Lines: | ||
** Controls: DH5-alpha (not transformed) | |||
** FHUA PHIE6 --> IPTG | ** FHUA PHIE6 --> IPTG | ||
** CPX --> AHL | ** CPX --> AHL | ||
*Media: | *Media: | ||
Line 47: | Line 88: | ||
**PBS + Tween + BSA | **PBS + Tween + BSA | ||
*Protocol | |||
#Add AHL/IPTG to Liquid Culture | #Add AHL/IPTG to Liquid Culture | ||
#Wait an hour for AHL induced expression | #Wait an hour for AHL induced expression | ||
#Incubate wells in | #Incubate wells in wash media for 30 minutes on rocker at speed 5 | ||
#Centrifuge cells out of media | #Centrifuge cells out of Liquid Culture media | ||
#Resuspend in | #Resuspend in wash media | ||
#Add cells to appropriate well | #Add cells to appropriate well | ||
#Place on rocker for 1 hour at speed 5 | #Place on rocker for 1 hour at speed 5 | ||
#Aspirate out media in wells | #Aspirate/Pipet out media in wells | ||
#Resuspend in 150 ul media and wash for 15 minutes on rocker (repeat 2-3 times) |
Latest revision as of 12:28, 27 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 -- started at 3 PM
- 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
- Resuspend in 150 ul media and wash for 15 minutes on rocker (repeat 2-3 times)