Sequencing of construct
michela.deleidi, Pascale Baden
Abstract
This protocol describes sequencing of construct.
Attachments
Steps
PCR to amplify region of interest
| A | B |
|---|---|
| Master mix | 1x |
| H2O | 13.3 |
| 5x Colorless Reaction Buffer (Promega, #M3005) | 4 |
| 10mM dNTPs (ThermoFisher, #R0182) | 0.4 |
| Fw primer (CMV FW) | 0.6 |
| Rv primer (BGH RV) | 0.6 |
| GoTaq Polymerase (Promega, #M3005) | 0.1 |
| Σ | 19 µl |
Mix 19µL MM with 1µL DNA (50ng).
PCR Program
Sodium acetate precipitation
Add 50µL sodium acetate (1mL 3Molarity (M) Na Acetate (Carl Roth) + 24mL 100% EtOH (VWR Chemicals BDH Prolabo)) to 20µL PCR product.
Mix well and centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Add 100µL 70% EtOH onto pellet.
Centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Add 100µL 70% EtOH onto pellet.
Centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Centrifuge upside down max. 600rcf,4°C (top of sample down on tissue paper) to remove EtOH.
Add 15µL MilliQ-H2O to pellet and vortex 15-20 min (speed 0-1).
Sequencing-PCR
| A | B |
|---|---|
| 1x sample | |
| H2O | 5.3 µl |
| 5x Terminator Sequencing buffer | 3.3 µl |
| BigDye v3.1 (ThermoFisher, #4337455) | 1.4 µl |
| Σ | 10 µl |
Add 5µL MM.
Add 4µL DNA from step 13.
Add 1µL primer FW or RV.
Sequencing program
Add 25µL sodium acetate to 10µL PCR product from.
Mix well and centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Add 100µL 70% EtOH onto pellet.
Centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Add 100µL 70% EtOH onto pellet.
Centrifuge at 3200rcf,4°C.
Remove supernatant (pat plate on paper).
Centrifuge upside down max. 600rcf,4°C (top of sample down on tissue paper) to remove EtOH.
Add 15µL MilliQ-H2O to pellet and vortex 15-20min (speed 0-1).
Load Sequencing-plate
Add 10µL Hi-Di Formamide (Applied Biosystems).
7µL DNA after purification (Step 28).
Store in 4°C until sequencing.
