Difference between revisions of "Assembly Ladders"

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==Introduction==
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The Registry created a unique ladder to use with the various gels we run for assemblies. The ladder contains bands at the 4 most useful sizes, which are made by PCR. The detailed protocol for ladder preparation is available [[Assembly Ladder Protocol|here]].
  
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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:
 
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*3230 base pairs - This is the average size of the plasmid backbones
 
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*1000 base pairs
 
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*500 base pairs
 
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*100 base pairs
 
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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.
 
  
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==Parts==
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===Ladder Components===
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[[Image:070831.png|thumb|right|Two variations of the Assembly Ladder]]
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The ladder contains the following parts:
  
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The following parts will be included in the ladder:
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{| border="1"
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! Part !! Plasmid !! Primers Used !! Size after PCR
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|-
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| align="center" | pSB1AK3
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| align="center" | -
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| align="center" | Prefix-R and Suffix-F
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| align="center" | 3189 bp
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|-
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| align="center" | I5010
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| align="center" | pSB3K3
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| align="center" | Prefix-F and Suffix-R
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| align="center" | 998 bp
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|-
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| align="center" | I13027
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| align="center" | pSB1A2
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| align="center" | Prefix-F and Suffix-R
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| align="center" | 506 bp
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|-
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| align="center" | J32015
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| align="center" | pSB1AK3
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| align="center" | Prefix-F and Suffix-R
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| align="center" | 106 bp
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|}
  
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*pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
 
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*I5010 <small>in pSB3K3</small> - 998 bp after PCR with Prefix-F and Suffix-R
 
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*I13027 <small>in pSB1A2</small> - 506 after PCR with Prefix-F and Suffix-R
 
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*J32015 <small>in pSB1AK3</small> - 106 bp after PCR with Prefix-F and Suffix-R
 
  
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==Procedure==
 
  
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Procedure was provided by [[User:Meaganl|Meagan Lizarazo]]
 
  
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===PCR Reaction===
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===Versions===
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* 100 &mu;L reaction (do 2 reactions for each part)
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The ladder can be made in two different ways - so that the 4 bands are all the same molarity or all the same brightness. For assemblies, the Registry uses the equality molarity ladder, which each band at .9 GP/&mu;L. By loading 20&mu;L, the amount of DNA in each band is equal to the theoretical yield loaded from each assembly. This allows us to quickly judge the efficiency of the assembly.
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** 100 &mu;L PCR Supermix High Fidelity (Invitrogen)
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** 1.5 &mu;L of each 40 &mu;M primer (The proper primer depends on the part - see above for which primer to use with each part)
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** 1 &mu;L diluted template DNA (10 ng/&mu;L)
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* Initial denature 95&deg;C 5 min
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* 35 cycles
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** 94&deg;C 30 sec
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** 55&deg;C 30 sec
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** 68&deg;C  - 4:00 min for pSB1AK3, I5010, & J32015, 36 sec for I13027
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* Final extension 68&deg; 10 min
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* 4&deg;C forever
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===Post PCR Cleanup: Qiagen PCR Cleanup Kit===
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* Elimination of PCR enzymes and dNTPs is required
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* Use Qiagen's [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx QIAquick PCR Purification kit]
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** Combine 200&mu;L of PCR product with 1000&mu;L (5X) Buffer PB
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** Transfer 1st half (600&mu;L) to QIAquick spin column
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** Spin at 8000g 1 minute, reload the 600&mu;L flow-through, spin again, discard flow-through
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** Load 2nd half (600&mu;L) to same QIAquick spin column
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** Spin at 8000g 1 minute, reload, spin again, discard flow-through
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** Add 750&mu;L Buffer PE, spin 17900g 1 minute, discard flow-through
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** Spin again 17900g 3 minutes to dry
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** Transfer column to a clean 1.7 mL tube, add 30 &mu;L TE 10:1 (pH 8.0) heated to 50&deg;C, spin at 8000g 1 minute
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** Add a further 30&mu;l TE, spin again
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** Reload 60&mu;L to column, spin 8000g 5 minutes
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*Measure yield with Nanodrop, expect 200-400ng/&mu;L in 55&mu;L
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Latest revision as of 02:50, 6 September 2007

The Registry created a unique ladder to use with the various gels we run for assemblies. The ladder contains bands at the 4 most useful sizes, which are made by PCR. The detailed protocol for ladder preparation is available here.


Ladder Components

Two variations of the Assembly Ladder

The ladder contains the following parts:

Part Plasmid Primers Used Size after PCR
pSB1AK3 - Prefix-R and Suffix-F 3189 bp
I5010 pSB3K3 Prefix-F and Suffix-R 998 bp
I13027 pSB1A2 Prefix-F and Suffix-R 506 bp
J32015 pSB1AK3 Prefix-F and Suffix-R 106 bp



Versions

The ladder can be made in two different ways - so that the 4 bands are all the same molarity or all the same brightness. For assemblies, the Registry uses the equality molarity ladder, which each band at .9 GP/μL. By loading 20μL, the amount of DNA in each band is equal to the theoretical yield loaded from each assembly. This allows us to quickly judge the efficiency of the assembly.