Chapter 8 PCR Protocols

8.1 DreamTaq (Any Primers)

  • Location in thermocyclers
    1. Dependent on primers

8.1.1 Protocol Mixture (DreamTaq)

Reagents Per sample (Microliters) Total Mix (Microliters)
Buffer 14 14*n
Primers LROR 0.5 0.5*n
Primers LR5 0.5 0.5*n
H2O 9 9*n
Total mix 24 24*n

Add 1uL of DNA

8.1.2 Protocol Conditions (DreamTaq)

DEPENDENT ON PRIMERS

8.2 ITS Metabarcoding (ITS1-F/ITS2)

  • Location in thermocyclers
    1. WALLE: TLAB/ITSBC
    2. EVA: ITS1/ITSBC

8.2.1 Protocol Mixture (ITS1-F/ITS2)

With sample number (n)

Reagent Per sample (Microliters) Total Mix (Microliters) Order
Buffer 8 8*n
dNTP’s 1 1*n
Primers ITS1F 0.5 0.5*n
Primers ITS2 0.5 0.5*n
Taq Polymerase 0.25 0.25*n LAST THING
H2O 13.75 13.75*n
Total mix 24 24*n

Add 1uL of DNA

8.2.2 Protocol Conditions (ITS1-F/ITS2)

Temperature Time Repeat
94 °C 1 min
95 °C 30 s x35
52 °C 30 s x35
72°C 30 s x35
72 °C 5 min
4 °C hold

8.3 ITS1/ITS4

  • Location in thermocyclers
    1. WALLE: ITS
    2. EVA: ITS

8.3.1 Protocol Mixture (ITS1/ITS4)

Reagents Per sample (Microliters) Total Mix (Microliters)
Buffer 8 8*n
dNTP’s 1 1*n
Primers ITS1 0.5 0.5*n
Primers ITS4 0.5 0.5*n
Taq Polymerase 0.25 0.25*n
H2O 9 9*n
Total mix 19.25 19.25*n

Add 1uL of DNA

8.3.2 Protocol Conditions (ITS1/ITS4)

8.4 LSU

  • Location in thermocyclers
    1. WALLE: LSU
    2. EVA: LSU

8.4.1 Protocol Mixture (LSU)

Reagents Per sample (Microliters) Total Mix (Microliters) Order
Buffer 2.5 2.5*n
dNTP’s 0.5 0.5*n
Primers LROR 1.25 1.25*n
Primers LR5 1.25 1.25*n
Taq Polymerase 0.25 0.25*n LAST THING
H2O 18.5 18.5*n
Total mix 24.25 24.25*n

Add 1uL of DNA

8.4.2 Protocol Conditions (LSU)

Temperature Time Repeat
94 °C 2 min
94 °C 1 min x35
50 °C 30 s x35
72 °C 1m 30s x35
72 °C 5 m
4 °C hold

8.5 BASF611/BASR1340

  • Location in thermocycler
    1. WALLE:
    2. EVA:

8.5.1 Protocol Mixture (BAS)

Reagents Per sample (Microliters) Total Mix (Microliters) Order
Buffer 8 8*n
dNTP’s 1 1*n
BasF (10uM) 0.5 0.5*n
BasR (10uM) 0.5 0.5*n
Taq Polymerase 0.25 0.25*n LAST THING
H2O 8.75 8.75*n
Total mix 19 19*n

Add 1uL of DNA

8.5.2 Protocol Conditions (BAS)

8.6 EXOSAP

  • Location in thermocycler
    1. WALLE: EXO
    2. EVA: EXO

8.6.1 Protocol Mixture (EXOSAP)

Reagent Per sample (Microliters) Total Mix per sample (Microliters)
ExoSAP 2 2
DNA 5 1

8.6.2 Protocol Conditions (EXOSAP)

Temperature Time Repeat
37 °C 15 min No
80 °C 15 min No

8.7 Primer sequences

8.7.1 16s for bacteria

FWD GTGYCAGCMGCCGCGGTAA

REV GGACTACNVGGGTWTCTAAT

8.7.2 LSU

LROR ACCCGCTGAACTTAAGC

LR5 TCCTGAGGGAAACTTCG

8.7.3 ITS1 (metabarcoding)

ITS1-F CTTGGTCATTTAGAGGAAGTAA

ITS2 GCTGCGTTCTTCATCGATGC

8.7.4 ITS whole region

ITS1 TCCGTAGGTGAACCTGCGG

ITS4 TCCTCCGCTTATTGATATGC