Date:

1 A 2 3 4

Plate number:
5 Actin Sample B 6 Actin Sample B 7 8 9 10 11 NTC 12 NTC

Actin Actin Sample Actin Sample Actin Sample A A A Sample B

Actin Actin Actin Sample C Sample C Sample C

Col1A1 Sample A

Col1A1 Sample A

Col1A1 Sample A

Col1A1 Sample B

Col1A1 Sample B

Col1A1 Sample B

Col1A1 Col1A1 Col1A1 Sample C Sample C Sample C

NTC

NTC

TGF-a Sample A

TGF-a Sample A

TGF-a Sample A

TGF-a Sample B

TGF-a Sample B

TGF-a Sample B

TGF-a TGF-a TGF-a Sample C Sample C Sample C

NTC

NTC

qPCR SYBR green protocol and calculator

4ng/ul cDNA stock preparation Fill in or calculate cDNA ng/ul cDNA Stock concentration Sample name ug/20ul cDNA ug/ul cDNA ng/ul cDNA dillution factor for 4ng/ul 1 well extra per primer take from take from add add cDNA H2O Total ul cDNA stock (ul) DEPC (ul) needed stock (ul) water (ul)

v1.1

Sample name

Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 Sample11 Sample12

0.5 0.5 0.2 2 2 2 2 2 2 2 2 2

0.025 0.025 0.01 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

25 25 10 100 100 100 100 100 100 100 100 100 (2)

6.25 6.25 2.5 25 25 25 25 25 25 25 25 25

1 1 1 1 1 1 1 1 1 1 1 1

5.25 5.25 1.5 24 24 24 24 24 24 24 24 24

44 44 44 44 44 44 44 44 44 44 44 44

7.0 7.0 17.6 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 (3)

37.0 37.0 26.4 42.2 42.2 42.2 42.2 42.2 42.2 42.2 42.2 42.2

Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7 Sample8 Sample9 Sample10 Sample11 Sample12

3 wells extra

ul/well

Primer name number of wells

actin 11 60

COl1A1 11 60

TGF-a 11 60

5 10

Primer number of wells Primers B Cybr needed cDNA (ul) from 4ng/ul in well

actin 14 70 140

COl1A1 14 70 140

TGF-a 14 70 140 (4) (5)

If marked in red double the amount of primers B preparation (150ul is not enough)

up to 5ul/well
120 120 120

(6)

cDNA ng/well

20

20

adjust cDNA to DDW 5ul

(7) 4 0 0
total volume in well 20ul

Prepare primers according to (1) add 15ul to well (from 4+5 mix)

Fill in cDNA concentration in (2)

prepare 4ng/ul cDNA stock according to (3)

Mix (4)+(5)

add cDNA from 4ng/ul stock to well according to (6)

If needed, add water according to (7)

Primers preparetion (dillution from 100uM to 500nM ( final concentration in well) 20ul primer F +20ul primer R 160ul DDW Depc Primers A + Primers B 30ul from primers A (1) * NTC = No cDNA sample, use water instead * SYBR Green Power SYBR green master mix (Applied biosystems) + 120ul DDW Depc

In microtube In microtube