Bridges Lab Protocols - User contributions [en]

PCR Analysis of Tail DNA

2016-02-17T18:43:26Z

Lmcallan: /* Protocol */

see [[Genotyping Details]] for strain specific details

==Materials==
# Dream Taq Green master mix
# Specific gene Primers (0.4um Working stock)
# Tail digest DNA
# ddH2O

==Protocol==
First, make 250ul of 0.4um working stock primers from 100um Primary Stocks.
#248 ul ddH20
#1ul forward primer (100um)
#1ul reverse primer (100um)

Use the following Volumes per 25ul Reaction:

Per sample (1X)
#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer)
#0.4um Primer Mix: 5ul
#Sterile ddH2O: 7.5ul

*Template: 1 uL

Run "specfic" PCR Program for gene of interest (approx 2 hours).

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2016-02-17T18:39:09Z

Lmcallan: /* Materials */

see [[Genotyping Details]] for strain specific details

==Materials==
# Dream Taq Green master mix
# Specific gene Primers (0.4um Working stock)
# Tail digest DNA
# ddH2O

==Protocol==
Use the following Volumes per 25ul Reaction:

Per sample 1X
#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#Sterile ddH2O: 7.5ul

*Template: 1 uL

Run "specfic" PCR Program for gene of interest (approx 2 hours).

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2016-02-17T18:37:16Z

Lmcallan:

see [[Genotyping Details]] for strain specific details

==Materials==
# Dream Taq Green master mix
# Specific Primers
# DNA
# ddH2O

==Protocol==
Use the following Volumes per 25ul Reaction:

Per sample 1X
#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#Sterile ddH2O: 7.5ul

*Template: 1 uL

Run "specfic" PCR Program for gene of interest (approx 2 hours).

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2016-02-17T18:36:39Z

Lmcallan: /* Protocol */

see [[Genotyping Details]] for strain specific details

==Materials==
# Dream Taq Green master mix
# Specific Primers
# DNA
# ddH2O

==Protocol==
Use the following Volumes per 25ul Reaction:

Per sample 1X
#Dream Tag Master mix: 12.5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#Sterile ddH2O: 7.5ul

*Template: 1 uL

Run "specfic" PCR Program for gene of interest (approx 2 hours).
* specific to each gene

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2016-02-17T18:29:28Z

Lmcallan: /* Materials */

see [[Genotyping Details]] for strain specific details

==Materials==
# Dream Taq Green master mix
# Specific Primers
# DNA
# ddH2O

==Protocol==
Use the following Volumes per 50ul Reaction:

#10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 29ul
#Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
#Template: 1 uL

Master Mix (Per 5mL -- Make 1mL Aliquots)
#10X GoTaq Buffer: 625uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 625ul
#dNTPs: 62.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 3625ul
#Polymerase Go-Taq: 15.625ul ("Molecular Biology Stuff" box in freezer)
*Add Template Individually

Run PCR Program (approx 2 hours).
Use Cycler 1 on 6th Floor
*Login: Sergey, Just press enter to Login
*Under Genotype folder, pick Ingles program for Ingles genotyping
*Under Genotype folder, pick regpcr program for PLT genotyping

Make sure to press enter 2x once to confirm Tubes and second time to start PCR

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

PCR Analysis of Tail DNA

2016-02-17T18:28:52Z

Lmcallan: /* Materials */

see [[Genotyping Details]] for strain specific details

==Materials==
Dream Taq Green master mix
Specific Primers
DNA
ddH2O

==Protocol==
Use the following Volumes per 50ul Reaction:

#10X GoTaq Buffer: 5uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 5ul
#dNTPs: 0.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 29ul
#Polymerase Go-Taq: 0.125uL ("Molecular Biology Stuff" box in freezer)
#Template: 1 uL

Master Mix (Per 5mL -- Make 1mL Aliquots)
#10X GoTaq Buffer: 625uL ("Molecular Biology Stuff" box in freezer)
#Primer Mix: 625ul
#dNTPs: 62.5uL of 10 mM ("Molecular Biology Stuff" box in freezer)
#Sterile water: 3625ul
#Polymerase Go-Taq: 15.625ul ("Molecular Biology Stuff" box in freezer)
*Add Template Individually

Run PCR Program (approx 2 hours).
Use Cycler 1 on 6th Floor
*Login: Sergey, Just press enter to Login
*Under Genotype folder, pick Ingles program for Ingles genotyping
*Under Genotype folder, pick regpcr program for PLT genotyping

Make sure to press enter 2x once to confirm Tubes and second time to start PCR

see [[Preparing an Agarose Gel]] for details on preparing a DNA gel

[[Category: Genotyping]]
[[Category: Mouse Work]]

Preparation of Tail Samples (for Genotyping)

2016-02-17T18:24:29Z

Lmcallan: /* Protocol */

==PBND Solution: Tail Lysis Buffer==
* 50mM KCl
* 10mM Tris HCl (pH~8.3)
* 0.1 mg/mL MgCl2, 6H20
* 0.1 mg/mL Gelatin
* 0.45% (NP-40) Jgepal
* 0.45% Tween 20

==Protocol==
# Combine 100uL of PBND solution with 2ul of Proteinase K and mouse tail in an eppendorf tube or in a well of a 96 well PCR plate (Proteinase K stock = 10mg/mL)
# Incubate at 55 degrees (16 hours - O/N)
# Incubate at 85 degrees for 60 min
# Hold at 4 degrees

[[Category: Mouse Work]]
[[Category: Molecular Biology]]
[[Category: DNA]]
[[Category: Genotyping]]

TaqMan SNP Assay

2015-11-16T16:39:59Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to 20ng/ul approx.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X): see table below).
# Add Reaction Mix (5.25 ul) added to each well (Table 1.)
# Diluted gDNA samples each added to a single well (Table 1.)

'''Table 1. Full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
|gDNA Sample (20ng/ul)
|4.50 ul
|501.6 ul
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:39:40Z

Lmcallan:

TaqMan SNP Assay

2015-11-16T16:38:48Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to 20ng/ul approx.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X)).
# Add Reaction Mix (5.25 ul) added to each well (Table 1.)
# Diluted gDNA samples each added to a single well (Table 1.)

'''Table 1. Full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
|gDNA Sample (20ng/ul)
|4.50
|501.6
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:38:21Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to 20ng/ul approx.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X)).
# Add Reaction Mix (5.25 ul) added to each well (Table 1.)
# Diluted gDNA samples each added to a single well (Table 1.)

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
|gDNA Sample (20ng/ul)
|4.50
|501.6
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:35:19Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).
# Add Reaction Mix (5.25 ul) added to each well (see Table 2.)
# Diluted gDNA samples each added to a single well (see Table 2.)

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul
|}

'''Table 2. for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|Reaction Master Mix
|5.25 ul
|554.4 ul
|-
|gDNA samples
|4.75 ul
|501.6 ul
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:34:34Z

Lmcallan: /* Protocol */

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul
|}

# Reaction Mix (5.25 ul) added to each well (see Table 2.)
# Diluted gDNA samples each added to a single well (see Table 2.)

'''Table 2. for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|Reaction Master Mix
|5.25 ul
|554.4 ul
|-
|gDNA samples
|4.75 ul
|501.6 ul
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:29:58Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul
|}

# Reaction Mix (5.25 ul) added to each well (see Table 2.)
# Diluted gDNA samples each added to a single well (see Table 2.)

'''Table 2. for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|Reaction Master Mix
|5.25 ul
|554.4 ul
|-
|gDNA samples
|4.75 ul
|501.6 ul
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:29:27Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul
|}

# Reaction Mix (5.25 ul) added to each well (see Table 2.)
# Diluted gDNA samples each added to a single well (see Table 2.)

'''Table 2. for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|Reaction Master Mix
|5.25 ul
|554.4 ul
|-
|gDNA samples
|4.75 ul
|501.6 ul
|-
!
!10 ul
!1056 ul
|}

TaqMan SNP Assay

2015-11-16T16:28:26Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

'''Table 1. Reaction Mix for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul

# Reaction Mix (5.25 ul) added to each well (see Table 2.)
# Diluted gDNA samples each added to a single well (see Table 2.)

'''Table 2. for full 96 well plate'''
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|Reaction Master Mix
|5.25 ul
|554.4 ul
|-
|gDNA samples
|4.75 ul
|501.6 ul
|-
!
!10 ul
!1056 ul

TaqMan SNP Assay

2015-11-16T16:20:09Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to 1-5 ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

Master Mix for full 96 well plate
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul

TaqMan SNP Assay

2015-11-16T16:19:59Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan® Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==
# Dilute gDNA samples to 1-5 ng/ul.
# Place gDNA, TaqMan® Genotyping Master Mix (40X), TaqMan® Genotyping Assay (2X) on ice.
# Make up Reaction Master mix (see table below).

Master Mix for full 96 well plate
{| class="wikitable"
|-
! Component
! 1X volume
! 96X Volume (+ 10% excess)
|-
|TaqMan® Genotyping Master Mix (2X)
|5.00 ul
|528.0 ul
|-
|TaqMan® Genotyping Assay (40X)
|0.25 ul
|26.4 ul
|-
!
!5.25 ul
!554.4 ul

TaqMan SNP Assay

2015-11-16T16:18:37Z

Lmcallan:

TaqMan SNP Assay

2015-11-16T16:18:03Z

Lmcallan:

TaqMan SNP Assay

2015-11-16T16:15:52Z

Lmcallan:

TaqMan SNP Assay

2015-11-16T16:14:58Z

Lmcallan:

TaqMan SNP Assay

2015-11-16T16:14:15Z

Lmcallan:

TaqMan SNP Assay

2015-11-12T17:21:13Z

Lmcallan:

TaqMan SNP Assay

2015-11-12T17:21:05Z

Lmcallan:

TaqMan SNP Assay

2015-11-12T17:20:41Z

Lmcallan:

TaqMan SNP Assay

2015-11-11T16:16:55Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==

TaqMan SNP Assay

2015-11-11T16:16:32Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described [[Blood DNA extraction]])
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==

TaqMan SNP Assay

2015-11-11T16:15:43Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA (extracted as described )
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==

TaqMan SNP Assay

2015-11-11T16:13:14Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==

TaqMan SNP Assay

2015-11-11T16:05:01Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed.
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*Nuclease-Free Water
*Sample Genomic DNA
*Postive control(s) (Minor allele) (''optional, but recommended'')

== Protocol ==

TaqMan SNP Assay

2015-11-11T16:01:43Z

Lmcallan:

TaqMan SNP Assay

2015-11-11T15:57:09Z

Lmcallan:

== Materials ==

*TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).
*TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).
*MicroAmp Fast 96-Well Plates.
*Optical Plate Covers.
*StepOnePlus Real-Time PCR System (Applied Biosystems).
*PC/Laptop with StepOnePlus Software installed

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:55:57Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) (Applied Biosystems).

TaqMan Genotyping Master Mix (supplied at 2X)(Applied Biosystems).

MicroAmp Fast 96-Well Plates.

Optical Plate Covers.

StepOnePlus Real-Time PCR System (Applied Biosystems).

PC/Laptop with StepOnePlus Software installed

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:51:22Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X).

TaqMan Genotyping Master Mix (supplied at 2X).

MicroAmp Fast 96-Well Plates.

Optical Plate Covers.

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:51:03Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X).

TaqMan Genotyping Master Mix (supplied at 2X).

MicroAmp Fast 96-Well Plates.
Optical Plate Covers.

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:50:43Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X).
----

TaqMan Genotyping Master Mix (supplied at 2X).
----

MicroAmp Fast 96-Well Plates.
Optical Plate Covers.

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:49:32Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X).
TaqMan Genotyping Master Mix (supplied at 2X).
MicroAmp Fast 96-Well Plates.
Optical Plate Covers.

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:48:50Z

Lmcallan:

== Materials ==

TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X)
TaqMan Genotyping Master Mix (supplied at 2X)
MicroAmp Fast 96-Well plates
Optical Plate Covers

== Protocol ==

TaqMan SNP Assay

2015-11-11T15:47:09Z

Lmcallan: Created page with "Materials TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X) TaqMan Genotyping Master Mix (supplied at 2X) MicroAmp Fast 96-Well plates Optical plate covers Protoc..."

[[Materials]]
TaqMan® Genotyping Assay (supplied at 20X, 40X or 80X)
TaqMan Genotyping Master Mix (supplied at 2X)
MicroAmp Fast 96-Well plates
Optical plate covers

[[Protocol]]

Main Page

2015-11-11T15:30:26Z

Lmcallan: /* Genotyping */

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.uthsc.edu Bridges Lab Website]. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

__NOTOC__

==Protocol Categories==
See [[Special:Categories]] for as listing of protocols by category.

==Cell and Tissue Culture==
*[[Splitting Cells]]
*[[Generating DMSO Stocks for Cell Culture]]
*[[Differentiation of 3T3-L1 Cells]]
*[[Electroporation of 3T3-L1 Adipocytes]]
*[[Fugene Transfection of 293T/COS Cells]]
*[[Lipofectamine Mediated Knockdown]]
*[[Lipofectamine Plasmid Transfection]]
*[[3T3-L1 Adipocyte Fractionation]]
*[[Preparing Cell Lysates]]
*[[Glucose Uptake Assay]]
*[[Luciferase Assay]]
*[[Inositol Labeling and Lipid Extraction]]

==Cloning and Molecular Biology==
*[[Transformation of Bacteria]]
*[[Mutagenesis]]
*[[PCR Amplification of DNA]]
*[[Restriction Enzyme Based Cloning]]
**[[Restriction Enzyme Based Cloning - Ordering Primers]]
*[[TOPO Cloning]]
*[[Preparing an Agarose Gel]]
*[[Cesium Chloride Preparation of DNA]]
*[[Designing and Generating CRISPR-Cas Mutants]]

==Cell Biology==
*[[Immunofluoresence]]
**[[Colocalization]]
*[[FM4-64 Labeling of Yeast Cells]]

==Protein Analysis==
*[[Preparing a SDS-PAGE Gel]]
*[[Western Blotting]]
*[[Surface Plasmon Resonance - Protein Lipid Interactions]]
===Protein Quantification===
*[[Bradford Assay]]
*[[Quantification by Absorbance at 280nm]]
*[[Determining Percent Purity]]
*[[Using ImageJ to Quantify Bands]]

===Protein Purification===
*[[French Press]]
*[[Purification of GST Fusion Proteins]]
*[[Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)]]
*[[Preparation of DIG Labelled Probes]]
*[[Glycogen Synthase Assay]]
*[[Triglyceride Assay from Cells and Tissues]]
*[[Triglyceride Assay from Drosophila (German Method)]]

==Transcriptional Analysis==
*[[Real Time PCR From Cell Culture]]
*[[Harvesting RNA from Cells grown in monolayer]]
*[[Using Bioconductor To Analyse Beadarray Data]]
*[[Using Bioconductor To Analyse Microarray Data]]

==Mouse/Fly Protocols==
*[[Maternal Particulate Exposure Breeding]]

==Fly Stocks==
*[[Maintenance of 18C Fly Stocks]]
*[[Fly Food Protocol]]

===Genotyping===
*[[Ear Tagging and Tail Clipping]]
*[[DNA Preparation from Tail Clip]]
*[[PCR Analysis of Tail DNA]]
*[[Colony PCR]]
*[[BDNF PCR]]
*[[TaqMan SNP Assay]]

===Metabolic Measurements===
*[[Glucose Tolerance Test]]
*[[Insulin Tolerance Test]]
*[[Measuring Fasting Insulin]]
*[[Monitoring Food Intake]]

==Tissue Preparation==
*[[Harvesting Mouse Tissue]]
*[[Preparation of Protein Lysates from Mouse Tissues]]
*[[Preparation of RNA Samples from Mouse Tissues]]
*[[Paraffin Embedding of Tissue Samples]]
*[[H&E Staining of Mouse Tissue]]
**[[Adipocyte Cell Counting with ImageJ]]
*[[Perfusion of Mouse]]

==Media and Buffers==
===Yeast Media===
*[[YPD Media and Agar]]
*[[Yeast Selective Media and Agar]]
===Buffers===
====Lysis Buffers====
*[[KRBH Buffer]]
*[[RIPA Buffer]]
*[[CHAPS Lysis Buffer]]
====SDS-PAGE Buffers====
*[[SDS-PAGE Separating Gel Buffer]]
*[[SDS-PAGE Stacking Gel Buffer]]
*[[TORC1 Kinase Buffer]]
*[[Acrylamide Solution]]
====Other Buffers====
*[[KRBH Buffer]]
*[[MTORC1 Kinase Assay]]
*[[TORC1 Kinase Buffer]]

==Figure Generation and Data Management==
*[[Generation of Figures in Illustrator]]
*[[Using Bioconductor To Analyse Microarray Data]]

TaqMan SNP Array

2015-11-11T15:24:47Z

Lmcallan: Created page with "Materials Needed"

Materials Needed