User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/26: Difference between revisions
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* Non-specific amplification. Need to gel purify. | * Non-specific amplification. Need to gel purify. | ||
* Use 30 uL of each per lane for gel purification. | * Use 30 uL of each per lane for gel purification. | ||
'''CONTINUED: 5/27/15''' | |||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 1. DBN006 PCR || 0.002 || -19 || 2.0 | ||
| | |||
| 2. | |||
|} | |} | ||
** Concentration is weird. Use max amt. of DNA for subsequent steps | |||
* Use BsaI | * Digest & Dephosphorylate the Insert(s) | ||
** Use BsaI | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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* 25°C, ∞ | * 25°C, ∞ | ||
* The final concentration is | * The final concentration is ??? | ||
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| bgcolor=#cfcfcf | Rxn2 | | bgcolor=#cfcfcf | Rxn2 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 6.0 || --- | ||
|- | |- | ||
| Vector DNA || 3.0 || 3.0 | | Vector DNA || 3.0 || 3.0 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 10.0 || 10.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || --- || | | dH<sub>2</sub>O || --- || 6.0 | ||
|- | |- | ||
| || | | || 20.0 μL || 20.0 μL | ||
|} | |} | ||
* Incubate at RT/ 10 min. | |||
* Add to 50 μL DH5α-turbo; ice/ 5min. | |||
* Plate on 100 μg/mL amp agar | |||
Revision as of 16:25, 27 May 2015
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05/26/15
Ben - luc replacement donor plasmid
Bio-Rad: "Phusion" (block #)
CONCLUSION
Thermal cycler program: Note: In the Haynes lab, use the LabNet OptiMax Thermocycler, Program "AnOlig RD"
Rene - CRISPR PCR library PCR trial
1-12. Luc14 g034 - Lu34_AA01 - Lu34_AA12
Labnet: "GoTaq"
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