User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions
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* LCR Development - PCR MV10 bacbone | * LCR Development - PCR MV10 bacbone | ||
* gRNA cloning (Rene) | |||
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* 72°C, 10 min | * 72°C, 10 min | ||
* 4°C ∞ | * 4°C ∞ | ||
---- | |||
'''gRNA cloning - Rene''' | |||
* Follow the "Oligo annealing and cloning into backbone vectors - NEW" protocol | |||
Phosphorylate and anneal oligo pairs | |||
# | |||
# | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
|- | |||
| 100 μM Oligo 1 || 1.0 | |||
|- | |||
| 100 μM Oligo 2 || 1.0 | |||
|- | |||
| 10x T4 Lign buf (NEB) || 1.0 | |||
|- | |||
| T4 PNK (NEB) || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 6.5 | |||
|- | |||
| || 10.0 | |||
|} | |||
Bio-Rad Thermocycler | |||
* 37°C, 30 min | |||
* 95°C, 5 min | |||
* Ramp down to 25°C at 5°C/min | |||
Dilute the product(s) 1:250 | |||
* Add 2 μL product to 498 μL dH<sub>2</sub>O | |||
Digestion/ Ligation Reactions (Vector/ dsOligo Insert) | |||
# ###/### | |||
# ###/### | |||
# ###/### | |||
{| {{table}} cellspacing="3" <!-- Oligo annealing table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn | |||
|- | |||
| 100 ng Vector || ## | |||
|- | |||
| 1:250 dsOligo || 2.0 | |||
|- | |||
| 10x FD buf (NEB) || 2.0 | |||
|- | |||
| 10 mM DTT || 1.0 | |||
|- | |||
| 10 mM ATP || 1.0 | |||
|- | |||
| FastDigest BbsI || 1.0 | |||
|- | |||
| T7 DNA ligase || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || ## | |||
|- | |||
| || 20.0 | |||
|} | |||
Bio-Rad Thermocycler | |||
* 6x [37°C, 5 min; 23°C, 5 min] | |||
* 4°C, ∞ | |||
Transformation(s) | |||
* 2.0 μL ligation + 50 μL DH5α-turbo | |||
* Plate on 100 μg/mL amp | |||
Revision as of 14:38, 16 April 2015
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04/16/15
LCR Development
Measure conc.
Program: Phusion (block B)
gRNA cloning - Rene
Phosphorylate and anneal oligo pairs
Bio-Rad Thermocycler
|