Assembly Ladders

Revision as of 18:29, 8 August 2007 by SBurke (Talk | contribs) (→‎PCR Reaction)

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:

  • pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
  • P1010 - 991 bp after PCR with VR and VF2
  • S03582 - 508 bp after PCR with VR and VF2

*I'm still looking for a 100 bp part to include. Sam 14:28, 8 August 2007 (EDT)

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
  • 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, expect 200-400ng/μL in 55μL