Difference between revisions of "Assembly Ladders"
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*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 |
| − | * | + | *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 | |
| − | < | + | |
==Procedure== | ==Procedure== | ||
Revision as of 19:25, 27 August 2007
Contents
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