Team:Stanford/Protocols

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{{Template:Stanford_2010_Main_Menu}}
{{Template:Stanford_2010_Main_Menu}}
<div class="contents">
<div class="contents">
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{|align = center
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|__TOC__
 +
|}
-
==Our Protocols==
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==Digestion Recipe==
 +
{|
 +
|DNA: || align="right" | 2 ug || (calculate volume based off concentration obtained from nanodrop)
 +
|-
 +
|10x BSA: || align="right" | 5 uL || (if needed, check NEB website)
 +
|-
 +
|10x NEBuffer: || align="right" | 5 uL || (use [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp the NEB website] to find which buffer)
 +
|-
 +
|Each enzyme: || align="right" | 1 uL || (add last; it's the most expensive ingredient)
 +
|-
 +
|Sterile water: || align="right" | ~26 uL || (so that total volume is 50uL)
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|}
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*[[Team:Stanford/Protocols/HeatTransformProtocol|Heat-Shock Transformation]]
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==Ligation Recipe==
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{|
 +
|Vector DNA: || align="right" | 150 ng
 +
|-
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|5 - 20x insert DNA: || align="right" | __ ng || --> (5 to 20) * (insert bp / vector bp) * 150 see [http://bioinfo.clontech.com/infusion/molarRatio.do this calculator] for help
 +
|-
 +
|10x T4 ligase buffer: || align="right" | 2 uL ||
 +
|-
 +
|T4 ligase: || align="right" | 1 uL || (add last; it's the most expensive ingredient)
 +
|-
 +
|Sterile water: || align="right" | __ uL || (so that total volume is 20uL)
 +
|}
 +
 
 +
==PCR Assembly==
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{|
 +
| Forward piece || align="right" | 5 uL
 +
|-
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| Backward piece || align="right" | 5 uL
 +
|-
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| PCR Supermix || align="right" | 40 uL
 +
|}
 +
 
 +
==Electroporation Transformation==
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'''Cells:''' E. coli DH10B
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'''Protocol:'''
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#Get DNA (0.5 to 2.0 uL)
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#Transfer DNA to electrocompetent cells (keep cells in ice bucket)
 +
#Resuspend cells and DNA
 +
#Transfer cells and DNA to cuvette (don't leave air bubbles, and hold cuvette at the top to keep the bottom cool)
 +
#Zap the cells (2000V, 25uF, 200ohms)
 +
#Add 1000uL SOC to cuvette, resuspend the cells, and transfer to microfuge tube
 +
#Incubate cells at 37oC for 30 to 60 minutes. Warm up agar plates in anticipation.
 +
#Centrifuge the cells, remove 800uL of supernatant, resuspend cells in remaining 200uL.
 +
#Plate the cells, incubate at 37oC for 12-16 hours.
 +
 
 +
==Heat-Shock Transformation==
 +
'''Cells:''' E. coli DH5alpha
 +
 
 +
'''Protocol:'''
 +
#Take out competent E.coli cells from –80 C freezer.
 +
#Turn on water bath to 42 C.
 +
#Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar) – you may need more or less cells, depending how competent they are.
 +
#Keep tubes on ice.
 +
#Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
 +
#Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
 +
#Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
 +
#Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
 +
#Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
 +
#Pick the colonies about 12-16 hours later.
</div>
</div>

Latest revision as of 20:19, 11 August 2010

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Contents

Digestion Recipe

DNA: 2 ug (calculate volume based off concentration obtained from nanodrop)
10x BSA: 5 uL (if needed, check NEB website)
10x NEBuffer: 5 uL (use [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp the NEB website] to find which buffer)
Each enzyme: 1 uL (add last; it's the most expensive ingredient)
Sterile water: ~26 uL (so that total volume is 50uL)

Ligation Recipe

Vector DNA: 150 ng
5 - 20x insert DNA: __ ng --> (5 to 20) * (insert bp / vector bp) * 150 see [http://bioinfo.clontech.com/infusion/molarRatio.do this calculator] for help
10x T4 ligase buffer: 2 uL
T4 ligase: 1 uL (add last; it's the most expensive ingredient)
Sterile water: __ uL (so that total volume is 20uL)

PCR Assembly

Forward piece 5 uL
Backward piece 5 uL
PCR Supermix 40 uL

Electroporation Transformation

Cells: E. coli DH10B

Protocol:

  1. Get DNA (0.5 to 2.0 uL)
  2. Transfer DNA to electrocompetent cells (keep cells in ice bucket)
  3. Resuspend cells and DNA
  4. Transfer cells and DNA to cuvette (don't leave air bubbles, and hold cuvette at the top to keep the bottom cool)
  5. Zap the cells (2000V, 25uF, 200ohms)
  6. Add 1000uL SOC to cuvette, resuspend the cells, and transfer to microfuge tube
  7. Incubate cells at 37oC for 30 to 60 minutes. Warm up agar plates in anticipation.
  8. Centrifuge the cells, remove 800uL of supernatant, resuspend cells in remaining 200uL.
  9. Plate the cells, incubate at 37oC for 12-16 hours.

Heat-Shock Transformation

Cells: E. coli DH5alpha

Protocol:

  1. Take out competent E.coli cells from –80 C freezer.
  2. Turn on water bath to 42 C.
  3. Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar) – you may need more or less cells, depending how competent they are.
  4. Keep tubes on ice.
  5. Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
  6. Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
  7. Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
  8. Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
  9. Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
  10. Pick the colonies about 12-16 hours later.
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