Assembly Ladders

Revision as of 18:55, 23 July 2007 by SBurke (Talk | contribs) (→‎Parts)

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 (Part G00100, 20 bp) and VR (Part G00101, 20 bp), 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