Difference between revisions of "Assembly Ladders"

(Parts)
(Parts)
Line 13: Line 13:
  
 
*pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
 
*pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
*I5010 - 998 bp after PCR with Prefix-F and Suffix-R
+
*I5010 <small>in pSB3K3</small> - 998 bp after PCR with Prefix-F and Suffix-R
*J32015 - 106 bp after PCR with Prefix-F and Suffix-R
+
*I13027 <small>in pSB1A2</small> - 506 after PCR with Prefix-F and Suffix-R
 
+
*J32015 <small>in pSB1AK3</small> - 106 bp after PCR with Prefix-F and Suffix-R
<nowiki>*</nowiki>I'm still looking for a 500 bp part to include. [[User:SBurke|Sam]] 14:38, 10 August 2007 (EDT)
+
  
 
==Procedure==
 
==Procedure==

Revision as of 19:25, 27 August 2007

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
  • 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
  • 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