Date_____ 1 A Col1a1 100ng 2 Col1a1 100ng 3 Col1a1 100ng 4 20

R____ 5 20 6 20 7 4 8 4 9 4 10 0.8 11 0.8 12 0.8

0.16

0.16

0.16

NTC

NTC

GAPDH 100ng

GAPDH 100ng

GAPDH 100ng

20

20

20

0.8

0.8

0.8

0.16

0.16

0.16

NTC

NTC

qPCR standard curve plate protocol and calculator

Sample name A B C cDNA Stock ug/20ul cDNA ug/ul 0.2 0.01 0.5 0.025 2 0.1 cDNA ng/ul 10 25 100

v1.2

If using cDNA from cDNA Add DDW 5 20

100ng/ul

cDNA stock 5 20 5 20 5 20 5 20 (1

Tube conc.

100ng/ul

20ng/ul

4ng/ul

0.8ng/ul

0.16ug/ul

0.032ng/ul

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 + 120ul DDW Depc
1 well Fill Primers names Col1a1 number of wells 15 GAPDH 15 15 15 5 10

In microtube In microtube
3 wells extra

(2

Primer number of wells Primers B Cybr

Col1a1 GAPDH 18 90 180 18 90 180 180 360

Total Primers B Total Cybr Green

Dillute cDNA sample according to (1) Mix primers B + Sybr Green according to (3) * wells are marked by total cDNA amount in well For 100ng/well * Use 5 dillutions for each primer * NTC = No cDNA sample, use water instead * SYBR Green Power SYBR green master mix (Applied biosystems) Prepare primers B according to (2) Add to each well 5ul from (1) add 5ul of 20ng/ul cDNA from (1)

(3

Add to each well 15ul from (3)