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# PCR Protocols
## DreamTaq (Any Primers)
- **Location in thermocyclers**
1. Dependent on primers
### 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***
### Protocol Conditions (DreamTaq)
**DEPENDENT ON PRIMERS**
## ITS Metabarcoding (ITS1-F/ITS2)
- **Location in thermocyclers**
1. WALLE: TLAB/ITSBC
2. EVA: ITS1/ITSBC
### 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***
### 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 | |
## ITS1/ITS4
- **Location in thermocyclers**
1. WALLE: ITS
2. EVA: ITS
### 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***
### Protocol Conditions (ITS1/ITS4)
## LSU
- **Location in thermocyclers**
1. WALLE: LSU
2. EVA: LSU
### 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***
### 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 | |
## BASF611/BASR1340
- Location in thermocycler
1. WALLE:
2. EVA:
### 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***
### Protocol Conditions (BAS)
## EXOSAP
- Location in thermocycler
1. WALLE: EXO
2. EVA: EXO
### Protocol Mixture (EXOSAP)
| Reagent | Per sample (Microliters) | Total Mix per sample (Microliters) |
|---------|--------------------------|------------------------------------|
| ExoSAP | 2 | 2 |
| DNA | 5 | 1 |
### Protocol Conditions (EXOSAP)
| Temperature | Time | Repeat |
|:-----------:|:------:|:------:|
| 37 °C | 15 min | No |
| 80 °C | 15 min | No |
***
## Primer sequences
### 16s for bacteria
>FWD
GTGYCAGCMGCCGCGGTAA
>REV
GGACTACNVGGGTWTCTAAT
### LSU
>LROR
ACCCGCTGAACTTAAGC
>LR5
TCCTGAGGGAAACTTCG
### ITS1 (metabarcoding)
>ITS1-F
CTTGGTCATTTAGAGGAAGTAA
>ITS2
GCTGCGTTCTTCATCGATGC
### ITS whole region
>ITS1
TCCGTAGGTGAACCTGCGG
>ITS4
TCCTCCGCTTATTGATATGC