Bridges Lab Protocols - User contributions [en]

CPY Assay

2010-08-12T19:57:11Z

Kalefish:

Carboxypeptidase Secretion Assay

== Protocol ==
*Revive Yeast Cells (2-3) days
*Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast on selective plates, allow to grow for 1 day. (24°C)
*Place nitrocellulose filters on gel and incubate overnight at 23°C. (Make sure to roll the sheet with a tube)
*Wash filter in water.
*Block filter with BSA for 1 hour.
*Wash in primary antibody overnight at 4C or >3h at room temp
(10,000x dilution)
*Rinse in Tbst.
*Secondary antibody at 10 000X for 45min-1h.
*Rinse in TBST.
*Develop

GoTaq PCR Genotyping

2010-08-10T16:21:33Z

Kalefish:

==Materials==
*GoTaq Master Mix
*Primer Mix (100uL Primers): Add 10uL of forward and reverse primer, 20uL total, into 1mL of dH20.
*Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, 14uL Nuclease-Free Water, and 1uL of template
*Gel: 0.3g Agarose to 30mL of TAE, microwave until dissolved and add 1uL of EtBr.

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==Procedure==
*Prepare gel 30 minutes before PCR is finished.
*Prepare Reaction Mixture, adding 13x all materials except for the template.
*Add this mixture to each PCR tube.
*Label and add each template to the corresponding PCR tube.
*Run PCR under genotyping>inoki (~1 hour)
*Load samples in gel and run on 30V (horizontally.)

GoTaq PCR Genotyping

2010-08-10T16:21:08Z

Kalefish: Created page with '==Materials(100uM)== *GoTaq Master Mix *Primer Mix: Add 10uL of forward and reverse primer, 20uL total, into 1mL of dH20. *Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, ...'

==Materials(100uM)==
*GoTaq Master Mix
*Primer Mix: Add 10uL of forward and reverse primer, 20uL total, into 1mL of dH20.
*Reaction Mixture: 25uL GoTaq Mix, 10uL of Primer Mix, 14uL Nuclease-Free Water, and 1uL of template
*Gel: 0.3g Agarose to 30mL of TAE, microwave until dissolved and add 1uL of EtBr.

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
==Procedure==
*Prepare gel 30 minutes before PCR is finished.
*Prepare Reaction Mixture, adding 13x all materials except for the template.
*Add this mixture to each PCR tube.
*Label and add each template to the corresponding PCR tube.
*Run PCR under genotyping>inoki (~1 hour)
*Load samples in gel and run on 30V (horizontally.)

CPY Assay

2010-07-08T14:15:08Z

Kalefish: /* Protocol */

Carboxypeptidase Secretion Assay

== Protocol ==
*Revive Yeast Cells (2-3) days
*Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast on selective plates, allow to grow for 1 day. (24°C)
*Place nitrocellulose filters on gel and incubate overnight at 23°C.
*Wash filter in water.
*Wash in primary antibody overnight at 4C or >3h at room temp
*Rinse in Tbst.
*Secondary antibody at 10 000X for 45min-1h.
*Rinse in TBST.
*Develop

CPY Assay

2010-07-07T16:38:30Z

Kalefish: /* Protocol */

Carboxypeptidase Secretion Assay

== Protocol ==
*Revive Yeast Cells (2-3) days
*Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast. (Does this have to be selective plates? or are YPD plates always used?)
*Place nitrocellulose filters on gel and incubate overnight at 23(So you're not actually moving the patch to the filter, but just allowing it to touch?)
*Wash filter in water (by adding water to dish and rocking?)
*Primary antibody (polyclonal against cpy location?) how long?
*Secondary antibody (where/what is it and how long?)
Wash with pbs in between?

What is ECL?

CPY Assay

2010-07-07T16:35:58Z

Kalefish: Created page with 'Carboxypeptidase Secretion Assay == Protocol == *Revive Yeast Cells (2-3) days *Pick one colony and make a 1/2 inch x 1/2 inch square patch of yeast. (Does this have to be sele...'

Dilution Formula

2010-06-29T13:46:17Z

Kalefish:

C1V1=C2V2

*C1=measured absorption
*V1=unknown volume to be added to final volume
*V2=desired volume to re-suspend V1 in (usually 4mL)
*C2=OD/2^x

*x=amount of times cells will double
*find this by dividing the number of hours the cells will be growing by how long one doubling period is

Dilution Formula

2010-06-29T13:45:22Z

Kalefish: Created page with 'C1V1=C2V2 C1=measured absorption V1=unknown volume to be added to final volume V2=desired volume to re-suspend V1 in (usually 4mL) C2=<math>OD/2^x</math> x=amount of times ce...'

C1V1=C2V2

C1=measured absorption
V1=unknown volume to be added to final volume
V2=desired volume to re-suspend V1 in (usually 4mL)
C2=<math>OD/2^x</math>
x=amount of times cells will double
^find this by dividing the number of hours the cells will be growing by how long one doubling period is

GST-EEA1 Pulldown from Yeast

2010-06-28T18:47:44Z

Kalefish:

== '''Materials''' ==
*2x HNG (100mL). Combine 100mM HEPES (10mL), 300mM NaCl (7.5mL), 20% glycerol (20mL)
*1x HNG (10mL). Mix 5mL 2xHNG and 1 PI tablet.
*Lysis Buffer (10mL). Combine 5mL 1x HNG, 1 PI tablet, 1mL of 1% NP40, and 50µL of magnesium chloride.
*Glutathione sepharose beads
*Glass beads

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== '''Protocol''' ==
*Inoculate cells in appropriate media overnight.
*Re-suspend cells in 1mL of lysis buffer.
*Lyse cells: place approximately 1mL of glass beads into sonicator tube (Weisman Lab) and run in the sonicator 3x for 20 seconds. (Invert tube in between each round)
*Centrifuge at 4°C for 10 minutes.
*Add 1.5mL of 1x HNG to GST and GST-EEA1 proteins. (-80°C)
*Combine 450µL of protein with 450µL of lysates.
*Place tubes end over end for 30 minutes at 4°C.
*Add 50µL of glutathione sepharose beads to each tube.
*Wash each tube with 1x HNG five times at 4°C.
*Load in a 4-12% gel.