Mar 30, 2023

Sequencing of construct

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4jy7jlo5/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.3byl4jy7jlo5/v1
Protocol Citation: Pascale Baden, michela.deleidi 2023. Sequencing of construct. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jy7jlo5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 79541
Keywords: Sequencing-PCR, Sodium acetate precipitation, Load Sequencing-plate, ASAPCRN, construct, construct this protocol, protocol
Abstract
This protocol describes sequencing of construct.
Attachments
Icon for 404-874.docx
404-874.docx
52KB
Materials
Materials


Fw primer (CMV FW)
Rv primer (BGH RV)
sodium acetate (Carl Roth)
EtOH (VWR Chemicals BDH Prolabo)
Terminator Sequencing buffer
MilliQ-H2O
Hi-Di Formamide (Applied Biosystems)
GoTaq(R) DNA Polymerase, 500uPromegaCatalog #M3005
dNTP Set 100 mM SolutionsThermo Fisher ScientificCatalog #R0182
BigDye™ Terminator v3.1 Cycle Sequencing KitThermo FisherCatalog #4337455

PCR to amplify region of interest
AB
Master mix1x
H2O13.3
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
Σ19 µl
Mix 19 µL MM with 1 µL DNA (50 ng ).

PCR Program


Sodium acetate precipitation
Add 50 µL sodium acetate (1 mL 3 Mass Percent Na Acetate (Carl Roth) + 24 mL 100% EtOH (VWR Chemicals BDH Prolabo)) to 20 µL PCR product.

Mix well and centrifuge at 3200 rcf, 4°C, 00:45:00 .
45m
Remove supernatant (pat plate on paper).
Add 100 µL 70% EtOH onto pellet.

Centrifuge at 3200 rcf, 4°C, 00:15:00 .

15m
Remove supernatant (pat plate on paper).
Add 100 µL 70% EtOH onto pellet.

Centrifuge at 3200 rcf, 4°C, 00:15:00 .
15m
Remove supernatant (pat plate on paper).
Centrifuge upside down max. 600 rcf, 4°C, 00:01:00 (top of sample down on tissue paper) to remove EtOH.

1m
Add 15 µL MilliQ-H2O to pellet and vortex 15-20 min (speed 0-1).

Sequencing-PCR
AB
1x sample
H2O5.3 µl
5x Terminator Sequencing buffer3.3 µl
BigDye v3.1 (ThermoFisher, #4337455)1.4 µl
Σ10 µl
Add 5 µL MM.
Add 4 µL DNA from step 13.

Add 1 µL primer FW or RV.

Sequencing program


Sodium acetate precipitation
Add 25 µL sodium acetate to 10 µL PCR product from.

Mix well and centrifuge at 3200 rcf, 4°C, 00:45:00 .

45m
Remove supernatant (pat plate on paper).
Add 100 µL 70% EtOH onto pellet.

Centrifuge at 3200 rcf, 4°C, 00:15:00 .
15m
Remove supernatant (pat plate on paper).
Add 100 µL 70% EtOH onto pellet.

Centrifuge at 3200 rcf, 4°C, 00:15:00 .
15m
Remove supernatant (pat plate on paper).
Centrifuge upside down max. 600 rcf, 4°C, 00:01:00 (top of sample down on tissue paper) to remove EtOH.

1m
Add 15 µL MilliQ-H2O to pellet and vortex 15-20min (speed 0-1).
Note
Note: COVER! Light sensitive!

Load Sequencing-plate
Add 10 µL Hi-Di Formamide (Applied Biosystems).

7 µL DNA after purification (Step 28).

Store in 4 °C until sequencing.