Assembly Ladders

Revision as of 18:50, 23 July 2007 by SBurke (Talk | contribs)

Introduction

We want to create a unique DNA ladder to use with the gels we run on assembly digests. The ladder will contain bands that are approximately the following sizes:

  • 3230 base pairs - This is the average size of the plasmid backbones
  • 1000 base pairs
  • 500 base pairs
  • 100 base pairs

Each band will also be at a particular concentration such that the relative amounts of DNA in each assembly digest can be compared to the expected amount.

Parts

The following parts will be included in the ladder:

  • I5010 - 958 base pairs (998 bp after PCR)*
  • J61026 - 448 base pairs (488 bp after PCR)
  • R1062 - 56 base pairs (96 bp after PCR)

*The primers, VF2 and VR, add 40 base pairs to each part during PCR.

Procedure

Procedure was written by Meagan Lizarazo

PCR Reaction

  • 100 μl reaction
    • 100 μl PCR Supermix High Fidelity (Invitrogen)
    • 1.5 μl VF2 primer (40 μM)
    • 1.5 μl VR primer (40 μM)
    • 1 μl diluted template DNA (10 ng/μl)


  • Cycle 35x
  • initial denature 95° 5 min
  • 35 cycles
    • 94° 30 sec
    • 55° 30 sec
    • 68° 4:00 min
  • final extension 68° 10 min
  • 4° 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°, spin at 8000g 1 minute
    • Add a further 30μl TE, spin again
    • Reload 60μl to column, spin 8000g 5 minutes
  • Measure yield with Nanodrop, expect 200-400ng/μl in 55μl