IGEM:IMPERIAL/2006/Protocols/S01656new: Difference between revisions
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==New | ==New Protocol to test aiiA Production== | ||
===Outline=== | ===Outline=== | ||
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*Wait 7 hrs | *Wait 7 hrs | ||
*'''6pm''' | *'''6pm''' | ||
**Test | **Test Fluorescence | ||
**Spin down cells + collect supernatant | **Spin down cells + collect supernatant | ||
*'''The Next Day''' - Test frozen supernatant for AHL with T9002 | *'''The Next Day''' - Test frozen supernatant for AHL with T9002 | ||
*This will show you that aiiA is being produced and that the cells | *This will show you that aiiA is being produced and that the cells fluoresce. | ||
*This can be easily adapted to | *This can be easily adapted to measure AHL conc every hour so that you get a curve. However we will not need to do this for this aiiA part as the aiiA gene is untagged and may have different properties to the aiiA we will actually be using This test is solely to see if this gene works. | ||
==Detailed | ==Detailed Protocol== | ||
===Equipment/Materials=== | ===Equipment/Materials=== | ||
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* 96 well plate | * 96 well plate | ||
=== | ===Protocol=== | ||
*'''Grow cultures''' of S01656 (4ml) and B0034 (2ml) overnight | *'''Grow cultures''' of S01656 (4ml) and B0034 (2ml) overnight | ||
*'''Make Fresh Day Cultures ''(10ml)''''' - Make '''2''' S01656 cultures and '''one''' B0034 culture they should have an optical density of '''0.1''' and you should use | *'''Make Fresh Day Cultures ''(10ml)''''' - Make '''2''' S01656 cultures and '''one''' B0034 culture they should have an optical density of '''0.1''' and you should use Pre-warmed media to prevent shock. Remember that the S01656 grows in kanomycin media. | ||
*'''You Should now have three cultures''' | *'''You Should now have three cultures''' | ||
*'''Wait''' for 2 hours | *'''Wait''' for 2 hours | ||
*'''Add AHL to eppindorf tubes (seven | *'''Add AHL to eppindorf tubes (seven different concentrations)''' Use the AHL dilution series below | ||
<br> | <br> | ||
{| border="1" cellpadding="1" | {| border="1" cellpadding="1" | ||
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|} | |} | ||
<br> | <br> | ||
*You should add 10ul of AHL to each eppindorf tube and 0.99ml of culture to each eppindorf in such a way that you have 21 tubes and each cell type has 7 tubes of | *You should add 10ul of AHL to each eppindorf tube and 0.99ml of culture to each eppindorf in such a way that you have 21 tubes and each cell type has 7 tubes of different AHL concentrations | ||
*'''Add 50ul of IPTG ''' to the + IPTG group of S01656 to induce aiiA production''' | *'''Add 50ul of IPTG ''' to the + IPTG group of S01656 to induce aiiA production''' | ||
*'''Wait''' for 7 hours | *'''Wait''' for 7 hours | ||
*'''Pipette into a 96 well plate''' And | *'''Pipette into a 96 well plate''' And measure the fluorescence using the wallic victor | ||
*'''put back into eppindorfs and | *'''put back into eppindorfs and centrifuge down cells | ||
*'''Pipette off supernatant and | *'''Pipette off supernatant and freeze''' | ||
*Tomorrow use T9002 | *Tomorrow use T9002 Protocol to measure amount of AHL |
Revision as of 03:40, 29 August 2006
New Protocol to test aiiA Production
Outline
- 9am - Make fresh day culture
- 11am - Add known AHL concs + IPTG
- Wait 7 hrs
- 6pm
- Test Fluorescence
- Spin down cells + collect supernatant
- The Next Day - Test frozen supernatant for AHL with T9002
- This will show you that aiiA is being produced and that the cells fluoresce.
- This can be easily adapted to measure AHL conc every hour so that you get a curve. However we will not need to do this for this aiiA part as the aiiA gene is untagged and may have different properties to the aiiA we will actually be using This test is solely to see if this gene works.
Detailed Protocol
Equipment/Materials
- Falcon tubes or similar
- Photospectrometer
- Gilson pipettes
- Eppendorf tubes
- Centrifuge
- Plain LB medium
- LB medium with 50 μg/mL Kanamycin
- LB medium with 50 μg/mL Ampicillin
- E. coli DH5a strain with part S01656
- E. coli DH5a strain with part T9002
- Perkin Elmer Victor 3 Fluorimeter
- 96 well plate
Protocol
- Grow cultures of S01656 (4ml) and B0034 (2ml) overnight
- Make Fresh Day Cultures (10ml) - Make 2 S01656 cultures and one B0034 culture they should have an optical density of 0.1 and you should use Pre-warmed media to prevent shock. Remember that the S01656 grows in kanomycin media.
- You Should now have three cultures
- Wait for 2 hours
- Add AHL to eppindorf tubes (seven different concentrations) Use the AHL dilution series below
|
- You should add 10ul of AHL to each eppindorf tube and 0.99ml of culture to each eppindorf in such a way that you have 21 tubes and each cell type has 7 tubes of different AHL concentrations
- Add 50ul of IPTG to the + IPTG group of S01656 to induce aiiA production
- Wait for 7 hours
- Pipette into a 96 well plate And measure the fluorescence using the wallic victor
- put back into eppindorfs and centrifuge down cells
- Pipette off supernatant and freeze
- Tomorrow use T9002 Protocol to measure amount of AHL