Difference between revisions of "Assembly Ladder Protocol"

 
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==Overview==
 
==Overview==
  
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The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes.For a description of the bands and why they were included, please visit the [[Assembly Ladders|Assembly Ladder Main Page]].  
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The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes. For a description of the bands and why they were included, please visit the [[Assembly Ladders|Assembly Ladder Main Page]].
  
 
==Materials==
 
==Materials==
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*PCR Supermix High Fidelity from Invitrogen
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*Prefix-F, Suffix-R, Prefix-R, and Suffix-F primers
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*Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification Kit]
  
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===Parts===
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*pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
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*I5010 <small>in pSB3K3</small> - 998 bp after PCR with Prefix-F and Suffix-R
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*I13027 <small>in pSB1A2</small> - 506 after PCR with Prefix-F and Suffix-R
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*J32015 <small>in pSB1AK3</small> - 106 bp after PCR with Prefix-F and Suffix-R
  
 
==Procedure==
 
==Procedure==
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*Measure yield with Nanodrop.
 
*Measure yield with Nanodrop.
  
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===Mixing the Ladder===
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==Contact==
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*Use one of the two following spreadsheets to determine what volumes are needed are needed for each part.
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*Who has experience with this protocol?
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**[https://parts.igem.org/wiki/index.php/Image:Equal_Molarity_Calculations.xls Bands at Equal Molarity]
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**[https://parts.igem.org/wiki/index.php/Image:Equal_Weight_Calculations.xls Bands at Equal Weight/Brightness]
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
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Latest revision as of 19:42, 31 August 2007

Overview

The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes. For a description of the bands and why they were included, please visit the Assembly Ladder Main Page.

Materials

  • PCR Supermix High Fidelity from Invitrogen
  • Prefix-F, Suffix-R, Prefix-R, and Suffix-F primers
  • Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification Kit]

Parts

  • pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
  • I5010 in pSB3K3 - 998 bp after PCR with Prefix-F and Suffix-R
  • I13027 in pSB1A2 - 506 after PCR with Prefix-F and Suffix-R
  • J32015 in pSB1AK3 - 106 bp after PCR with Prefix-F and Suffix-R

Procedure

Procedure was provided by Meagan Lizarazo

PCR Reaction

  • 100 μL reaction (do 2 reactions for each part)
    • 100 μL PCR Supermix High Fidelity (Invitrogen)
    • 1.5 μL of each 40 μM primer (The proper primer depends on the part - see above for which primer to use with each part)
    • 1 μL diluted template DNA (10 ng/μL)


  • Initial denature 95°C 5 min
  • 35 cycles
    • 94°C 30 sec
    • 55°C 30 sec
    • 68°C - 4:00 min for pSB1AK3, I5010, & J32015, 36 sec for I13027
  • Final extension 68° 10 min
  • 4°C forever

Post PCR Cleanup: Qiagen PCR Cleanup Kit

  • Elimination of PCR enzymes and dNTPs is required
  • Use Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification kit]
    • Combine 200μL of PCR product with 1000μL (5X) Buffer PB
    • Transfer 1st half (600μL) to QIAquick spin column
    • Spin at 8000g 1 minute, reload the 600μL flow-through, spin again, discard flow-through
    • Load 2nd half (600μL) to same QIAquick spin column
    • Spin at 8000g 1 minute, reload, spin again, discard flow-through
    • Add 750μL Buffer PE, spin 17900g 1 minute, discard flow-through
    • Spin again 17900g 3 minutes to dry
    • Transfer column to a clean 1.7 mL tube, add 30 μL TE 10:1 (pH 8.0) heated to 50°C, spin at 8000g 1 minute
    • Add a further 30μl TE, spin again
    • Reload 60μL to column, spin 8000g 5 minutes
  • Measure yield with Nanodrop.

Mixing the Ladder