|
|
| Line 1: |
Line 1: |
| − | ==Introduction==
| + | 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]]. |
| | | | |
| − | 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== | + | ===Ladder Components=== |
| | + | [[Image:070831.png|thumb|right|Two variations of the Assembly Ladder]] |
| | + | The ladder contains the following parts: |
| | | | |
| − | The following parts will be included in the ladder:
| + | {| border="1" |
| − | | + | ! Part !! Plasmid !! Primers Used !! Size after PCR |
| − | *pSB1AK3 - 3189 bp, PCR with Prefix-R and Suffix-F
| + | |- |
| − | *I5010 <small>in pSB3K3</small> - 998 bp after PCR with Prefix-F and Suffix-R
| + | | align="center" | pSB1AK3 |
| − | *I13027 <small>in pSB1A2</small> - 506 after PCR with Prefix-F and Suffix-R
| + | | align="center" | - |
| − | *J32015 <small>in pSB1AK3</small> - 106 bp after PCR with Prefix-F and Suffix-R
| + | | align="center" | Prefix-R and Suffix-F |
| − | | + | | align="center" | 3189 bp |
| − | ==Procedure== | + | |- |
| − | | + | | align="center" | I5010 |
| − | Procedure was provided by [[User:Meaganl|Meagan Lizarazo]]
| + | | align="center" | pSB3K3 |
| − | | + | | align="center" | Prefix-F and Suffix-R |
| − | ===PCR Reaction=== | + | | align="center" | 998 bp |
| − | * 100 μL reaction (do 2 reactions for each part)
| + | |- |
| − | ** 100 μL PCR Supermix High Fidelity (Invitrogen)
| + | | align="center" | I13027 |
| − | ** 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)
| + | | align="center" | pSB1A2 |
| − | ** 1 μL diluted template DNA (10 ng/μL)
| + | | align="center" | Prefix-F and Suffix-R |
| − | | + | | align="center" | 506 bp |
| − | | + | |- |
| − | * Initial denature 95°C 5 min
| + | | align="center" | J32015 |
| − | * 35 cycles
| + | | align="center" | pSB1AK3 |
| − | ** 94°C 30 sec
| + | | align="center" | Prefix-F and Suffix-R |
| − | ** 55°C 30 sec
| + | | align="center" | 106 bp |
| − | ** 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, expect 200-400ng/μL in 55μL
| + | |
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.
