05/27/15
- Rene - hi-res melt curve screen on CRISPR clones
- Ben - continue luc replacement donor cloning
Rene - hi-res melt curve screen on CRISPR clones
- Set-up
- 2 replicates per clone
- Total wells: 36 (1 well per rxn.)
- Template+water Multiplier = none (use 10x diluted stock for all rxns)
- Primer+SYBR Mix Multiplier = 27 + 3 = 39
- Template+ Water: Dilute PCR reaction 10x
- Use multichannel to transfer 5.0 μL PCR products into 45.0 μL dH2O
- Set up dilutions in 8-tube strips
- Use 4.5 μL Template+water per rxn
- Lu34_AA01 - 12
- Ga34_AA01 - 12
- nTc (3 wells)
- Master Mix: Primers+SYBR
- F = P149
- R = P160
- 10.5 per well
| Reagent |
Volume |
(x30)
|
| 750 nM F/R primers |
3.0 |
90.0
|
| 2x SYBR MM |
7.5 |
225
|
| |
15.0
|
- Plate loading
- Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
- Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
- Pipette 10.5 Primers+SYBR into C1-3 (nTc wells)
- Multi-channel pipette 4.5 Template+water into appropriate wells (A1-12, B1-12)
- Pipette 4.5 water into C1-3 (nTc wells)
- Run PCR - Roche LC480: "SYBR Karmella CNV melt curve Template"
- 95°C, 5 min
- 30x [95°C, 10 sec; 60°C, 10 sec; 72°C, 10 sec (measure)]
- Melt: continuous acquisition; 95°C, 4 sec; 70°C, 60 sec, -- +0.1°C/ 5 sec (programmed as "6 acquisitions/°C") --> 95°C
- Cooling: 40°C, 30 sec
RESULTS:
- Lu34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
- Ga34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C
- nTc (3 wells) -- no amplification
- Try using more diluted template (100x)
- Continue by sequencing a few purified PCR products with P215 to find a reference sample (wild type sequence)
|
Karmella's BioBrick Cloning
