Date: 10/10/2023 (am) Tuesday Date: 10/10/2023 (am) Tuesday

Task: To compare the results of Eppendorf thermal block and Benchmark thermal
block (both closed lid) of Thalassemia primer using Qiacube gDNA and swab at
temperature 65oC for 40 mins with 25 µl mineral oil and 0.5 ml tube.
Primer: Thalassemia primer
DNA: Qiacube AE, Qiacube H2O, swab, Blank
Task: To optimize Thalassemia primer using different mastermix and post or pre-
staining at temperature 65oC for 40 mins with 25 µl mineral oil and 0.5 ml tube.
Primer: Thalassemia primer
DNA: Qiacube AE, Qiacube H2O, swab, Blank
Thermal block: Benchmark

Preparation of PCR master mix Preparation of PCR master mix

Mastermix Mastermix
1X 9x 1X 5x
No Dye Isothermal 15.0 µl 135 µl No Dye Isothermal 15.0 µl 75 µl
F3 0.5 µl 4.5 µl F3 0.5 µl 2.5 µl
B3 0.5 µl 4.5 µl B3 0.5 µl 2.5 µl
FIP 1.0 µl 9 µl FIP 1.0 µl 5.0 µl
BIP 1.0 µl 9 µl BIP 1.0 µl 5.0 µl
DNA 2/0 µl Individual DNA 2/0 µl Individual
H2 O 5/7 µl Individual H2 O 5/7 µl Individual
Total vol 25 µl µl Total vol 25 µl µl

Aliquot 18µl mastermix into each tube. Aliquot 18 µl mastermix into each tube.

Aliquot 2µl DNA accordingly. Add H2O accordingly.

Add H2O up to 25µl. Add 25µl of mineral oil into each tube.

Add 25µl of mineral oil into each tube. Add 2µl DNA (poke through mineral oil) accordingly outside laminar flow.

PCR Reaction Vortex and centrifuge for a few seconds.

Run reaction in thermal block at temperature 65oC for 40 mins.
PCR Reaction
Add 1 ul of 1000X Sybr green dye after finishing the PCR reaction. Run reaction in thermal block at temperature 65oC for 40 mins.

Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction.
Date: 11/10/2023 (am) Wednesday Date: 11/10/2023 (am) Wednesday

Task: To optimize Thalassemia primer using post or pre-staining at temperature 65oC Task: To optimize Thalassemia primer using 0.5 ml tube and 0.2 ml strip tube on
for 40 mins with 25 µl mineral oil and 0.5 ml tube. Eppendorf thermal block at temperature 65oC for 40 mins with 25 µl mineral oil.

Primer: Thalassemia primer Primer: Thalassemia primer

DNA: Qiacube AE, Qiacube H2O, swab, Blank DNA: Qiacube AE, Qiacube H2O, swab, Blank
Thermal block: Benchmark Thermal block: Eppendorf

Preparation of PCR master mix Preparation of PCR master mix

Mastermix Mastermix
1X 6x 1X 11x
No Dye Isothermal 15.0 µl 90 µl No Dye Isothermal 15.0 µl 165 µl
F3 0.5 µl 3.0 µl F3 0.5 µl 5.5 µl
B3 0.5 µl 3.0 µl B3 0.5 µl 5.5 µl
FIP 1.0 µl 6.0 µl FIP 1.0 µl 11.0 µl
BIP 1.0 µl 6.0 µl BIP 1.0 µl 11.0 µl
DNA 2/0 µl Individual DNA 2/0 µl Individual
H2 O 5/7 µl Individual H2 O 5/7 µl Individual
Total vol 25 µl µl Total vol 25 µl µl

Aliquot 18 µl mastermix into each tube. Aliquot 18 µl mastermix into each tube.

Add H2O accordingly. Add H2O accordingly.

Add 25µl of mineral oil into each tube. Add 25µl of mineral oil into each tube.

Add 2µl DNA (poke through mineral oil) accordingly outside laminar flow. Add 2µl DNA (poke through mineral oil) accordingly outside laminar flow.

Vortex and centrifuge for a few seconds. Vortex and centrifuge for a few seconds.

PCR Reaction PCR Reaction

Run reaction in thermal block at temperature 65oC for 40 mins. Run reaction in thermal block at temperature 65oC for 40 mins.

Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction. Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction.
Date: 11/10/2023 (pm) Wednesday Date: 13/10/2023 (am) Friday
Task: To optimize Thalassemia primer using 0.5 ul helicase and 0.2 ml strip tube on Task: To optimize double mutation primer SMN1 H2O (FIP-mut/BIP-3B) without loop
Eppendorf thermal block at temperature 65oC for 40 mins with 25 µl mineral oil. using different helicase volume in non-template control on Eppendorf thermal block at
temperature 59oC for 30 mins with 25 µl mineral oil.
Primer: Thalassemia primer
DNA: Qiacube AE, Qiacube H2O, swab, Blank Primer: Thalassemia primer
Thermal block: Eppendorf DNA: Blank
Thermal block: Eppendorf
Preparation of PCR master mix
Preparation of PCR master mix
Mastermix
1X 7x Mastermix
No Dye Isothermal 15.0 µl 105 µl 1X 7x
F3 0.5 µl 3.5 µl No Dye Isothermal 15.0 µl 105 µl
B3 0.5 µl 3.5 µl Primer double mut SMN1 2.5 µl 17.5 µl
FIP 1.0 µl 7.0 µl H2 O µl Individual µl
BIP 1.0 µl 7.0 µl Helicase µl Individual µl
DNA 2/0 µl Individual
H2 O 5/7 µl Individual Aliquot 17.5 µl mastermix into each tube.
Total vol 25 µl µl
Add H2O accordingly.
Aliquot 18 µl mastermix into each tube. Add 25µl of mineral oil into each tube.
Add H2O accordingly. Vortex and centrifuge for a few seconds.
Add 25µl of mineral oil into each tube. PCR Reaction
Run reaction in thermal block at temperature 65oC for 40 mins.
Add 2µl DNA (poke through mineral oil) accordingly outside laminar flow.
Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction.
Vortex and centrifuge for a few seconds.

PCR Reaction
Run reaction in thermal block at temperature 65oC for 40 mins.

Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction.
Date: 13/10/2023 (am) Friday
Task: To optimize double mutation primer SMN1 H2O (FIP-mut/BIP-3B) without loop
using 0.2 ul helicase volume in 5 replicate non-template control on Eppendorf thermal
block at temperature 59oC for 30 mins with 25 µl mineral oil.

Primer: Thalassemia primer

DNA: Blank
Thermal block: Eppendorf

Preparation of PCR master mix

Mastermix
1X 6x
No Dye Isothermal 15.0 µl 90 µl
Primer double mut SMN1 2.5 µl 15 µl
H2 O 7.3 µl 43.8 µl
Helicase 0.2 µl 1.2 µl

Aliquot 25 µl mastermix into each tube.

Add H2O accordingly.

Add 25µl of mineral oil into each tube.

Vortex and centrifuge for a few seconds.

PCR Reaction
Run reaction in thermal block at temperature 65oC for 40 mins.

Add 1 ul of 1000X Sybr green dye (poke through mineral oil) after finishing the PCR reaction.