TITLE: DNA extraction and PCR amplification of petB gene of marine Synechococcus
AUTHORS: Denise Rui Ying Ong

[DESCRIPTION]
This protocol was described in the paper “Nested PCR Approach for petB Gene Metabarcoding of Sorted Marine Synechococcus Populations”. The objective was to create a nested PCR approach for amplifying the petB (encoding the cytochrome b6 subunit of the cytochrome b6f complex) marker gene from marine Synechococcus populations. The protocol can be used with DNA templates from samples obtained by traditional filtration methods as well as flow cytometry sorting, which often have low nucleic acid concentrations. We suggested that our protocol would be of interest to those applying flow cytometry for cell physiology measurements, such that these studies can also obtain the community composition simultaneously.

This protocol has two parts: 

1) The steps used to extract the DNA from filtered marine seawater samples and flow cytometry sorted samples.
2) Standard and nested PCR amplification reactions of the petB marker gene.


[STEPS]
SECTION: Primers
1. 
  
      Primer name  Sequence  Reference    petB-F  5'-TACGACTGGTTCCAGGAACG-3'  Mazard et al. (2012)    petB-R  5'-GAAGTGCATGAGCATGAA-3'  Mazard et al. (2012)    petB-50F  5'-CAGGACATYGCTGAY-3'  Ong et al. (submitted)    petB-634R  5'-GCTTVCGRATCATCARGAAG-3'  Ong et al. (submitted)  
 
SECTION: DNA extraction of filtered samples
2. 1.5 - 2L of seawater was filtered through a 0.22μm pore-size Sterivex filter.  DNA extraction from Sterivex filters require the following kits:
  
  

Refer to https://github.com/deniseong/marine-Synechococcus-metaB/tree/main/6_DNA%20extration%20protocol for step-by-step protocol.
SECTION: DNA extraction of flow cytometry sorted cells
4. Synechococcus cells from seawater samples were sorted through flow cytometry and collected in Eppendorf tubes containing Tris-EDTA lysis sorting buffer (10 mM Tris pH8.0, 1 mM EDTA pH8.0 and 1.2% Triton x-100).
SECTION: Nested PCR of filtered and sorted samples
20. Two sequential PCR reactions are conducted for nested PCR. A nested PCR is necessary to amplify the petB gene of flow cytometry sorted Synechococcus populations.

For the first PCR reaction, prepare the mastermix in a PCR clean microcentrifuge tube in UV hood.

Each sample is performed in duplicate reactions (2 x 10 μL). 
For each PCR, include at least one positive control and one negative control.

Each reaction is conducted to a final volume of 10 μL:
5 µL   
0.3 µL  (0.3 µM final concentration)
0.3 µL (0.3 µM final concentration)
0.05 µL (0.1 μg/μL final concentration)
DNA template (maximum 4µL of template, 0.54 ng/μL average final concentration for filtered samples or volume corresponding to approximately 160-400 sorted Synechococcus cells)
nuclease free water to a final volume of 10 μL per sample

Always prepare extra reactions, as some solution will be lost during pipetting. 

To calculate the total amount of mastermix volume per N samples, multiply above reaction volumes by X, whereby 

X = 2 x N + (number of positive controls) + (number of negative controls) + (number of spare reactions)
SECTION: Standard PCR of filtered samples
15. In UV hood, prepare the mastermix in a PCR clean microcentrifuge tube.

Each sample is performed in duplicate reactions (2 x 25 μL). 
For each PCR, include at least one positive control and one negative control.

Each reaction is conducted to a final volume of 25 μL:
12.5 µL   
0.75 µL  (0.3 µM final concentration)
0.75 µL (0.3 µM final concentration)
0.125 to 0.5 µL (0.1 to 0.4 μg/μL final concentration)
DNA template (maximum 10µL of template, 1.3 ng/μL average final concentration)
nuclease free water to a final volume of 25 μL per sample

Always prepare extra reactions, as some solution will be lost during pipetting. 

To calculate the total amount of mastermix volume per N samples, multiply above reaction volumnes by X, whereby 

X = 2 x N + (number of positive controls) + (number of negative controls) + (number of spare reactions)
SECTION: DNA extraction of flow cytometry sorted cells
5. Remove the tubes with cells from the -80 °C  and place them at a Room temperature  in a water bath.

SECTION: DNA extraction of flow cytometry sorted cells
6. Once thawed, place the tubes in   for a few seconds until frozen.

SECTION: DNA extraction of flow cytometry sorted cells
7. Repeat steps 5 and 6 two more times.
SECTION: DNA extraction of flow cytometry sorted cells
8. After DNA extraction, store samples in -80 °C until PCR.

SECTION: DNA extraction of filtered samples
3. After DNA extraction, store samples in -80 °C until PCR.

SECTION: Set-up before PCR
9. Clean all surfaces and pipettes with 70% ethanol before working.

Use a separate set of pipettes for loading mastermix in the UV hood and loading DNA template. Ideally, pipettes used to prepare mastermix stays in the UV hood and is never used for DNA. The other set stays on the bench.
SECTION: Set-up before PCR
10. Use filtered pipette tips to avoid contamination.
SECTION: Set-up before PCR
11. Prepare mastermix in UV hood. Place PCR clean microcentrifuge tubes, PCR clean plates, seals, pipettes, pipette tips and tube racks in the UV hood.

Clean with 70% ethanol and UV for 15 minutes. 
SECTION: Set-up before PCR
12. Store all PCR reagents (e.g. polymerase, primers, nuclease-free water, BSA)  at -20 °C until use. Before using all PCR reagents, ensure that the reagent is completely thawed. 
Only open PCR reagents in the UV hood. Use only filtered pipette tips to transfer the reagents. Ideally, PCR reagents are used in aliquots. 
SECTION: Set-up before PCR
13. Dilute primers to 10 µM in clean UV hood. 
For primers at 100µM, add 10µl of primer to 90µl of nuclease free water. 
SECTION: Standard PCR of filtered samples
16. Dispense mastermix into PCR wells in UV hood.

Add nuclease free water into negative controls inside UV hood.

Add DNA template of samples and postive controls outside UV hood.

Seal PCR plates/tubes and spin down.
SECTION: Standard PCR of filtered samples
17. Load PCR plates/tubes in thermocycler using the following thermal conditions:

      Temperature (°C)  Time  Cycles      94  5 min  1x  Initial Denaturation    94  30 s  30x  Denaturation    55  30 s  30x  Primer Annealing    72  45 s  30x  Extension    72  6 min  1x  Final Extension    4  ∞  1x  Hold  

Hold at 4 °C until collection of PCR plates/tubes.
SECTION: Standard PCR of filtered samples
18. Pool duplicate samples. 
Store in -20 °C .

SECTION: Standard PCR of filtered samples
19. Perform gel electrophoresis to check results of PCR.
SECTION: Nested PCR of filtered and sorted samples
21. Dispense mastermix into PCR wells in UV hood.

Add nuclease free water into negative controls inside UV hood.

Add DNA template of samples and postive controls outside UV hood.

Seal PCR plates/tubes and spin down.
SECTION: Nested PCR of filtered and sorted samples
22. Load PCR plates/tubes in thermocycler using the following thermal conditions:
 
      Temperature (°C)  Time  Cycles      94  5 min  1x  Initial Denaturation    94  30 s  30x  Denaturation    59  30 s  30x  Primer Annealing    72  45 s  30x  Extension    72  6 min  1x  Final Extension    4  ∞  1x  Hold  

Hold at 4 °C until collection of PCR plates/tubes.
SECTION: Nested PCR of filtered and sorted samples
23. Pool duplicate samples. 
Store at -20 °C .
SECTION: Nested PCR of filtered and sorted samples
24. For the second round of PCR reaction, prepare the mastermix in a PCR clean microcentrifuge tube in UV hood.

Each sample is performed in duplicate reactions (2 x 25 μL). 
For each PCR, include at least one positive control and one negative control.

Each reaction is conducted to a final volume of 25 μL:
12.5 µL   
0.75 µL  (0.3 µM final concentration)
0.75 µL (0.3 µM final concentration)
2.5 µL first round PCR product after pooling duplicates 
8.5 µL nuclease free water to a final volume of 10 μL per sample

Always prepare extra reactions, as some solution will be lost during pipetting. 

To calculate the total amount of mastermix volume per N samples, multiply above reaction volumes by X, whereby 

X = 2 x N + (number of positive controls) + (number of negative controls) + (number of spare reactions)

SECTION: Nested PCR of filtered and sorted samples
25. Dispense mastermix into PCR wells in UV hood.

Add nuclease free water into negative controls inside UV hood.

Add DNA template of samples and postive controls outside UV hood.

Seal PCR plates/tubes and spin down.
SECTION: Nested PCR of filtered and sorted samples
26. Load PCR plates/tubes in thermocycler using the following thermal conditions:

      Temperature (°C)  Time  Cycles      94  5 min  1x  Initial Denaturation    94  30 s  30x  Denaturation    55  30 s  30x  Primer Annealing    72  45 s  30x  Extension    72  6 min  1x  Final Extension    4  ∞  1x  Hold  

Hold at 4 °C until collection of PCR plates/tubes.
SECTION: Nested PCR of filtered and sorted samples
27. Pool duplicate samples. 
Store in -20 °C .
SECTION: Nested PCR of filtered and sorted samples
28. Perform gel electrophoresis to check results of PCR.
SECTION: Set-up before PCR
14. Ensure DNA template is completely thawed before adding to mastermix. 
To reduce DNA degradation in the sample, place the samples in an eppendorf tube rack over ice when loading DNA template.