16s, ITS2, 23s PCR Protocol Testing

20191119 R.S., E.S.

Protocol testing for 16s, ITS2, and 23s amplicon sequencing.

Primers: ITS2, cp23S, and 16S with Nextera partial tails

Primer     Annealing Temp
ITSintfor2 ITS2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGAATTGCAGAACTCCGTG 52°
ITS2_Reverse ITS2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGATCCATATGCTTAAGTTCAGCGGGT 52°
24 F cp23S-rDNA TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGTAGCAAATTTCCTTGTCG 55°
454 R cp23S-rDNA GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTGTATCGCTTTGTTCACC 55°
518F 16S V4V5 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCAGCAGCYGCGGTAAN 57°
926R 16S V4V5 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGTCAATTCNTTTRAGT, GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGTCAATTTCTTTGAGT, GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCGTCTATTCCTTTGANT 57°

Phusion HiFi Mastermix (Thermo Scientific™ F531S): https://www.fishersci.com/shop/products/phusion-high-fidelity-pcr-master-mixes/f531s

Protocol:

  1. Check concentration with Qubit.
  2. Dilute DNA to 100ng in 10ul (10ng/ul).
SampleID CoralCode Conc (ng/ul) DNA for 10ng/ul dilution (ul) Water for dilution (ul) Amount to add for 10ng Water to add Amount for mix
H1 1637H 49.6 2.02 7.98 1.00 47.00 0.167
H2 1060H 44.3 2.26 7.74 1.00 47.00 0.167
H3 1041H 44 2.27 7.73 1.00 47.00 0.167
H4 1651H 27 3.70 6.30 1.00 47.00 0.167
H5 1600H 26.6 3.76 6.24 1.00 47.00 0.167
H6 1196H 17.6 5.68 4.32 1.00 47.00 0.167
HM MixH 10     1.00 47.00  
S1 1637S 86.6 1.15 8.85 1.00 47.00 0.167
S2 1060S 71.8 1.39 8.61 1.00 47.00 0.167
S3 1041S 62.4 1.60 8.40 1.00 47.00 0.167
S4 1651S 24.5 4.08 5.92 1.00 47.00 0.167
S5 1600S 38.4 2.60 7.40 1.00 47.00 0.167
S6 1196S 25.6 3.91 6.09 1.00 47.00 0.167
SM MixS 10     1.00 47.00  
  1. Make stock solution per gene.
For 18 reactions (ul)        
Component Per Rxn ITS2 cp23S 16S
2X Phusion Mastermix 50ul 900 900 900
F primer (10uM) 3-4ul 72 72 54 (use mixed pool)
R primer (10uM) 3-4ul 72 72  
H2O Up to 100ul 738 738 828
  1. Add 99uL of stock solution to each well. Add 1ul of DNA to each well per gene.
  2. Aliquot 100ul reaction into 3 wells to run reaction in triplicate PCR (33ul each).
ITS2   cp23S   16S    
Cycles Time Temp Time Temp Time Temp
1 3 min 95° 3 min 95° 3 min 98°
35 30 sec 95° 30 sec 95° 30 sec 94°
  30 sec 52° 30 sec 55° 45 sec 57°
  30 sec 72° 30 sec 72° 1 min 72°
1 5 min 72° 5 min 72° 5 min 72°
1 ∞ min 4° ∞ min 4° ∞ min 4°
  1. Pool products into strip tubes (100ul total).
  2. Run 5ul of each product on a 2% agarose gel to check for ~300bp product.

Gel Electrophoresis Protocol

20191119 Gel

Order of test tubes, gel electrophoresis, and PCR plates:

Dilution Strip tubes:                
1 H1 H2 H3 H4 H5 H6 HM NegCon
2 S1 S2 S3 S4 S5 S6 SM NegCon
ITS2           cp23S            
  1 2 3 4 5 6 7 8 9 10 11 12
A H1 H1 H1 S1 S1 S1 H1 H1 H1 S1 S1 S1
B H2 H2 H2 S2 S2 S2 H2 H2 H2 S2 S2 S2
C H3 H3 H3 S3 S3 S3 H3 H3 H3 S3 S3 S3
D H4 H4 H4 S4 S4 S4 H4 H4 H4 S4 S4 S4
E H5 H5 H5 S5 S5 S5 H5 H5 H5 S5 S5 S5
F H6 H6 H6 S6 S6 S6 H6 H6 H6 S6 S6 S6
G HM HM HM SM SM SM HM HM HM SM SM SM
H NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG
V4V5 16S            
  13 14 15 16 17 18
A H1 H1 H1 S1 S1 S1
B H2 H2 H2 S2 S2 S2
C H3 H3 H3 S3 S3 S3
D H4 H4 H4 S4 S4 S4
E H5 H5 H5 S5 S5 S5
F H6 H6 H6 S6 S6 S6
G HM HM HM SM SM SM
H NEG NEG NEG NEG NEG NEG
PCR product strip tubes:                
1 H1-ITS2 H2-ITS2 H3-ITS2 H4-ITS2 H5-ITS2 H6-ITS2 HM-ITS2 NEG-ITS2
3 S1-ITS2 S2-ITS2 S3-ITS2 S4-ITS2 S5-ITS2 S6-ITS2 SM-ITS2 NEG-ITS2
7 H1-cp23S H2-cp23S H3-cp23S H4-cp23S H5-cp23S H6-cp23S HM-cp23S NEG-cp23S
10 S1-cp23S S2-cp23S S3-cp23S S4-cp23S S5-cp23S S6-cp23S SM-cp23S NEG-cp23S
13 H1-16S H2-16S H3-16S H4-16S H5-16S H6-16S HM-16S NEG-16S
16 S1-16S S2-16S S3-16S S4-16S S5-16S S6-16S SM-16S NEG-16S
Strip tubes submitted to Janet:                
1 H1-ITS2 H2-ITS2 H3-ITS2 H4-ITS2 H5-ITS2 H6-ITS2 HM-ITS2 S1-ITS2
2 S2-ITS2 S3-ITS2 S4-ITS2 S5-ITS2 S6-ITS2 SM-ITS2 H1-cp23S H2-cp23S
3 H3-cp23S H4-cp23S H5-cp23S H6-cp23S HM-cp23S S1-cp23S S2-cp23S S3-cp23S
4 S4-cp23S S5-cp23S S6-cp23S SM-cp23S H1-16S H2-16S H3-16S H4-16S
5 H5-16S H6-16S HM-16S S1-16S S2-16S S3-16S S4-16S S5-16S
6 S6-16S SM-16S            

Gel map:

Ladder H1-ITS2 H2-ITS2 H3-ITS2 H4-ITS2 H5-ITS2 H6-ITS2 HM-ITS2 NEG-ITS2 S1-ITS2 S2-ITS2 S3-ITS2 S4-ITS2 S5-ITS2 S6-ITS2 SM-ITS2 NEG-ITS2 H1-cp23S H2-cp23S H3-cp23S H4-cp23S H5-cp23S H6-cp23S HM-cp23S NEG-cp23S
                                                 
Ladder S1-cp23S S2-cp23S S3-cp23S S4-cp23S S5-cp23S S6-cp23S SM-cp23S NEG-cp23S H1-16S H2-16S H3-16S H4-16S H5-16S H6-16S HM-16S NEG-16S S1-16S S2-16S S3-16S S4-16S S5-16S S6-16S SM-16S NEG-16S

Final Amplicon samples:

SampleNumber SampleID CoralCode CoralType AmpliconGene ProductLength
1 H1 1637H Hard ITS2 350
2 H2 1060H Hard ITS2 350
3 H3 1041H Hard ITS2 350
4 H4 1651H Hard ITS2 350
5 H5 1600H Hard ITS2 350
6 H6 1196H Hard ITS2 350
7 HM MixH Hard ITS2 350
8 S1 1637S Soft ITS2 350
9 S2 1060S Soft ITS2 350
10 S3 1041S Soft ITS2 350
11 S4 1651S Soft ITS2 350
12 S5 1600S Soft ITS2 350
13 S6 1196S Soft ITS2 350
14 SM MixS Soft ITS2 350
15 H1 1637H Hard cp23S 350
16 H2 1060H Hard cp23S 350
17 H3 1041H Hard cp23S 350
18 H4 1651H Hard cp23S 350
19 H5 1600H Hard cp23S 350
20 H6 1196H Hard cp23S 350
21 HM MixH Hard cp23S 350
22 S1 1637S Soft cp23S 350
23 S2 1060S Soft cp23S 350
24 S3 1041S Soft cp23S 350
25 S4 1651S Soft cp23S 350
26 S5 1600S Soft cp23S 350
27 S6 1196S Soft cp23S 350
28 SM MixS Soft cp23S 350
29 H1 1637H Hard 16S 400
30 H2 1060H Hard 16S 400
31 H3 1041H Hard 16S 400
32 H4 1651H Hard 16S 400
33 H5 1600H Hard 16S 400
34 H6 1196H Hard 16S 400
35 HM MixH Hard 16S 400
36 S1 1637S Soft 16S 400
37 S2 1060S Soft 16S 400
38 S3 1041S Soft 16S 400
39 S4 1651S Soft 16S 400
40 S5 1600S Soft 16S 400
41 S6 1196S Soft 16S 400
42 SM MixS Soft 16S 400
Written on November 20, 2019