Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/30: Difference between revisions
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(Autocreate 2015/11/30 Entry for Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR) |
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==Overview== | ==Overview== | ||
Performing LCR on the PNK-treated parts from last week. | |||
==Methods== | |||
* Add components to the PNK DNA reaction as described in the table below | |||
* Set up the reactions in two PCR tubes, 25 µL per tube | |||
{| {{table}} | |||
| Reagent || Volume | |||
|- | |||
| PNK DNA || 20 μL | |||
|- | |||
| Oligo Bridges || 15.0 (3 each) | |||
|- | |||
| 10X Ampligase Buffer || 5.0 | |||
|- | |||
| DMSO || 4.0 | |||
|- | |||
| Betaine (5M) || 4.5 | |||
| Ampligase || 1.7 | |||
|- | |||
| dH<sub>2</sub>O || 0 μL | |||
|- | |||
| || 50.2 μL | |||
|} | |||
* Thermal cycler program | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Step''' | |||
| align="center" style="background:#f0f0f0;"|'''Temp (°C)''' | |||
| align="center" style="background:#f0f0f0;"|'''Time (s)''' | |||
|- | |||
| Initial Denaturation||94||120 | |||
|- | |||
| 50 cycles|||| | |||
|- | |||
| Denaturation||94||10 | |||
|- | |||
| Annealing||55||30 | |||
|- | |||
| Ligation||66||60 | |||
|- | |||
| |||| | |||
|- | |||
| Hold||4||∞ | |||
|} | |||
Revision as of 10:48, 30 November 2015
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OverviewPerforming LCR on the PNK-treated parts from last week. Methods
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