Assembly Ladder Protocol

Revision as of 18:25, 31 August 2007 by SBurke (Talk | contribs) (→‎Overview)

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
  • VR, VF2, Prefix-F, and Suffix-R 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