Difference between revisions of "Assembly Ladder Protocol"
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==Overview== | ==Overview== | ||
| − | The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes.For a description of the bands and why they were included, please visit the [[Assembly Ladders|Assembly Ladder Main Page]]. | + | The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes. For a description of the bands and why they were included, please visit the [[Assembly Ladders|Assembly Ladder Main Page]]. |
==Materials== | ==Materials== | ||
*PCR Supermix High Fidelity from Invitrogen | *PCR Supermix High Fidelity from Invitrogen | ||
| − | * | + | *Prefix-F, Suffix-R, Prefix-R, and Suffix-F primers |
| − | Qiagen's QIAquick PCR Purification Kit | + | *Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification Kit] |
===Parts=== | ===Parts=== | ||
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*Measure yield with Nanodrop. | *Measure yield with Nanodrop. | ||
| − | + | ===Mixing the Ladder=== | |
| − | == | + | *Use one of the two following spreadsheets to determine what volumes are needed are needed for each part. |
| − | * | + | **[https://parts.igem.org/wiki/index.php/Image:Equal_Molarity_Calculations.xls Bands at Equal Molarity] |
| − | + | **[https://parts.igem.org/wiki/index.php/Image:Equal_Weight_Calculations.xls Bands at Equal Weight/Brightness] | |
| − | + | ||
Latest revision as of 19:42, 31 August 2007
Contents
Overview
The Registry's Assembly DNA Ladder contains bands at what we consider the 4 most useful sizes. For a description of the bands and why they were included, please visit the Assembly Ladder Main Page.
Materials
- PCR Supermix High Fidelity from Invitrogen
- Prefix-F, Suffix-R, Prefix-R, and Suffix-F primers
- Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification Kit]
Parts
- 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 for pSB1AK3, I5010, & J32015, 36 sec for I13027
- 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.
Mixing the Ladder
- Use one of the two following spreadsheets to determine what volumes are needed are needed for each part.