Assembly Ladder Protocol

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

• Use one of the two following spreadsheets to determine what volumes are needed are needed for each part.
  • Bands at Equal Molarity
  • Bands at Equal Weight/Brightness

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.