Primer plates

Protocol 2 of 7. Comes after Part carrier. Next is Cargo ordering and pool preparation.

Order 74 primers as one 96-well plate, resuspend them to 100 µM, and split 1 working plate at 10 µM, one of them thawed for each run that needs these primers.

Primers: 74 in a 96-well plate
Stock plate: 100 µM
Working plates: 1 at 10 µM
Storage: -20 °C

Leaves you with

primer stock plate 74 primers seated in a 96-well plate · 100 µM
primer working plate 1 a copy of primer stock plate, used for one run · 10 µM, 100 µL per well

primer working plate 1: never return to the freezer a thawed working plate is used once and discarded

Materials

Name	Per run
nuclease-free water	25,160 µL in all
Empty 96-well plate	1 for the working plates
Adhesive plate seals	2 seals

Equipment: Multichannel pipette, Plate shaker, Plate centrifuge

Oligos

Order sheet: ../pool-primers.tsv

74 oligos · 20 bases

For	Names	Oligos	Wells
P1 batch forward	OP107	1	primer stock plate A1
P2 gene reverse	some of OP1 to OP80	72	primer stock plate, 72 wells from A2 to G2
P3 batch outer	OP146	1	primer stock plate A3

All 74 rows

Name	Sequence (5'→3')	Length	For
OP107	`CCGTGCGACAAGATTTCAAG`	20	P1 batch forward
OP1	`AAACACGTGGCAAACATTCC`	20	P2 gene reverse
OP146	`TACTTGAATACCACGTGGCC`	20	P3 batch outer
OP2	`AAACCGGAGCCATACAGTAC`	20	P2 gene reverse
OP3	`AAAGCACTCTTAGGCCTCTG`	20	P2 gene reverse
OP4	`AAAGGGGCCGTCAATATCAG`	20	P2 gene reverse
OP5	`AAATAAGACGACGACCCTCG`	20	P2 gene reverse
OP6	`AACGATGATGCTCACTCTCG`	20	P2 gene reverse
OP11	`AAGAATTACTGACCCCTCGG`	20	P2 gene reverse
OP12	`AAGACGATCCGAGCCATTAC`	20	P2 gene reverse
OP13	`AAGGAACTATGGCATCGAGC`	20	P2 gene reverse
OP14	`AAGGACTGCATACCAGGTTG`	20	P2 gene reverse
OP15	`AAGGATATGTAGACACCGCC`	20	P2 gene reverse
OP16	`AAGGCCCAGAAGGATACAAC`	20	P2 gene reverse
OP17	`AAGGCGCTCGGATAATACTC`	20	P2 gene reverse
OP18	`AAGGTATGTATAGCGACCGC`	20	P2 gene reverse
OP20	`AATAGGAACCTCTTACGCGG`	20	P2 gene reverse
OP21	`AATATCACGCAAAAGCACCG`	20	P2 gene reverse
OP22	`AATCAGTTTCTTTGGCAGCC`	20	P2 gene reverse
OP23	`AATGCAAAGCTATTAGCGCG`	20	P2 gene reverse
OP24	`AATGCGTCATTTTACACGGC`	20	P2 gene reverse
OP25	`AATGTCCTTAGGCAGTCGTC`	20	P2 gene reverse
OP26	`ACAACGAGCAGACCGAATAG`	20	P2 gene reverse
OP27	`ACAAGGAGTCGGCATATCAC`	20	P2 gene reverse
OP28	`ACAGAACGAACAGGCACTAC`	20	P2 gene reverse
OP29	`ACAGGAAGCAAGGTATACGC`	20	P2 gene reverse
OP30	`ACAGGGTATATTGAGTGCCC`	20	P2 gene reverse
OP31	`ACATAAGCGATCCCAAGGTC`	20	P2 gene reverse
OP32	`ACATCGCATACCAGAACAGG`	20	P2 gene reverse
OP33	`ACATTAAATTTCGCCGTGGC`	20	P2 gene reverse
OP34	`ACCACAGGTCAAGATTCACG`	20	P2 gene reverse
OP35	`ACCCGTATCGCATAAGGATG`	20	P2 gene reverse
OP36	`ACGAGATGATGCACCGATAG`	20	P2 gene reverse
OP37	`ACGATGGGGACATAGAACAC`	20	P2 gene reverse
OP38	`ACGGAGCCCTTATTGTAACC`	20	P2 gene reverse
OP39	`ACGTATGGGGAACACTACAC`	20	P2 gene reverse
OP40	`ACGTGAAACTGTATCGAGCC`	20	P2 gene reverse
OP41	`ACGTTCAGTTTTCCAATGGC`	20	P2 gene reverse
OP42	`ACTAGATTAGCAAGGCACCC`	20	P2 gene reverse
OP43	`ACTGGACCCAATAAAAGGCC`	20	P2 gene reverse
OP44	`ACTTCGATTGGCAAGGACTG`	20	P2 gene reverse
OP45	`AGAACATAGCATTCACGGGG`	20	P2 gene reverse
OP46	`AGACAACAATCTGAGGCTGG`	20	P2 gene reverse
OP47	`AGACAAGCCTTAACCGTAGG`	20	P2 gene reverse
OP48	`AGACACAAGGCTGATTCCAG`	20	P2 gene reverse
OP49	`AGACATGGGATTGACCACAC`	20	P2 gene reverse
OP50	`AGAGAGGCATGATTGACCTC`	20	P2 gene reverse
OP51	`AGAGTTGCACCTAGAATCCG`	20	P2 gene reverse
OP52	`AGATAGATGCTCCGTCAAGC`	20	P2 gene reverse
OP53	`AGATAGTCACGCACAAGACC`	20	P2 gene reverse
OP54	`AGATTAGCCGACTTTCCTGG`	20	P2 gene reverse
OP55	`AGATTAGCTGCCGATACTGG`	20	P2 gene reverse
OP56	`AGATTGTTACTCCGACGGAC`	20	P2 gene reverse
OP57	`AGATTTCCGACGAGATTCCC`	20	P2 gene reverse
OP58	`AGCATCCGTCTAAATCTCGG`	20	P2 gene reverse
OP59	`AGCTATAAGAATTGCCGGGC`	20	P2 gene reverse
OP60	`AGCTATGATCCCGGTGTAAC`	20	P2 gene reverse
OP61	`AGCTCAATCTAACAGTGGGG`	20	P2 gene reverse
OP62	`AGGACACCAGACCAATGAAG`	20	P2 gene reverse
OP63	`AGGGCTAATTACCATCAGCG`	20	P2 gene reverse
OP64	`AGGTGATCTGACGAATGTCC`	20	P2 gene reverse
OP65	`AGTAAAGCATAGTGCCCAGC`	20	P2 gene reverse
OP67	`AGTAGTATCCGAATCGCTGC`	20	P2 gene reverse
OP68	`AGTATCTCAGCAAGGGCAAC`	20	P2 gene reverse
OP69	`AGTATTAGGCGTCAAGGTCC`	20	P2 gene reverse
OP70	`AGTATTCTTACAGCCAGCCG`	20	P2 gene reverse
OP71	`AGTATTGCCGGACTAAACCC`	20	P2 gene reverse
OP73	`AGTCCCAAGTTCAGACGTAC`	20	P2 gene reverse
OP74	`AGTCCGACACAATGTGACAC`	20	P2 gene reverse
OP75	`AGTGAACTGACCGAATCCTC`	20	P2 gene reverse
OP77	`AGTGGTCTGTAAACCGTACC`	20	P2 gene reverse
OP78	`AGTGTTTTCCATTTTCCGCG`	20	P2 gene reverse
OP79	`AGTTATAAGGGTCCGATGCC`	20	P2 gene reverse
OP80	`AGTTGCAGTATCTAACCCGC`	20	P2 gene reverse

Plates

primer stock plate 96 wells · 74 kinds

primer working plate 1 96 wells · 74 kinds · 10 µM, 100 µL per well, -20 °C

1Order the primers

Order every primer on the oligo sheet at the top of this page as one dried 96-well plate.

Expected result

A sealed 96-well plate holding 74 dried primers.

2Resuspend the primers to 100 µM

Caution: Spin the plate down before taking the seal off.

Add 250 µL of nuclease-free water to each of the 74 wells.
Seal the plate, shake it until every pellet is in solution, and spin it down.

Expected result

Every well holds 250 µL at 100 µM.

3Seat the stock plate

Check the supplier's plate map against the primer stock plate map below, well by well.
Write primer stock plate and the date on the plate skirt.

Expected result

Every well of primer stock plate holds the primer the map below names.

4Split 1 working plate at 10 µM

primer working plate 1: never return to the freezer a thawed working plate is used once and discarded

Add 90 µL of nuclease-free water to the first 74 wells of each working plate.
Move 10 µL from each well of primer stock plate into the same well of every working plate.
Seal each plate, shake it, and spin it down.

Stock into primer working plate 1 74 moves · 10 µL each · each well into the same well

74 moves

From	To	µL
primer stock plate A1	primer working plate 1 A1	10
primer stock plate A2	primer working plate 1 A2	10
primer stock plate A3	primer working plate 1 A3	10
primer stock plate A4	primer working plate 1 A4	10
primer stock plate A5	primer working plate 1 A5	10
primer stock plate A6	primer working plate 1 A6	10
primer stock plate A7	primer working plate 1 A7	10
primer stock plate A8	primer working plate 1 A8	10
primer stock plate A9	primer working plate 1 A9	10
primer stock plate A10	primer working plate 1 A10	10
primer stock plate A11	primer working plate 1 A11	10
primer stock plate A12	primer working plate 1 A12	10
primer stock plate B1	primer working plate 1 B1	10
primer stock plate B2	primer working plate 1 B2	10
primer stock plate B3	primer working plate 1 B3	10
primer stock plate B4	primer working plate 1 B4	10
primer stock plate B5	primer working plate 1 B5	10
primer stock plate B6	primer working plate 1 B6	10
primer stock plate B7	primer working plate 1 B7	10
primer stock plate B8	primer working plate 1 B8	10
primer stock plate B9	primer working plate 1 B9	10
primer stock plate B10	primer working plate 1 B10	10
primer stock plate B11	primer working plate 1 B11	10
primer stock plate B12	primer working plate 1 B12	10
primer stock plate C1	primer working plate 1 C1	10
primer stock plate C2	primer working plate 1 C2	10
primer stock plate C3	primer working plate 1 C3	10
primer stock plate C4	primer working plate 1 C4	10
primer stock plate C5	primer working plate 1 C5	10
primer stock plate C6	primer working plate 1 C6	10
primer stock plate C7	primer working plate 1 C7	10
primer stock plate C8	primer working plate 1 C8	10
primer stock plate C9	primer working plate 1 C9	10
primer stock plate C10	primer working plate 1 C10	10
primer stock plate C11	primer working plate 1 C11	10
primer stock plate C12	primer working plate 1 C12	10
primer stock plate D1	primer working plate 1 D1	10
primer stock plate D2	primer working plate 1 D2	10
primer stock plate D3	primer working plate 1 D3	10
primer stock plate D4	primer working plate 1 D4	10
primer stock plate D5	primer working plate 1 D5	10
primer stock plate D6	primer working plate 1 D6	10
primer stock plate D7	primer working plate 1 D7	10
primer stock plate D8	primer working plate 1 D8	10
primer stock plate D9	primer working plate 1 D9	10
primer stock plate D10	primer working plate 1 D10	10
primer stock plate D11	primer working plate 1 D11	10
primer stock plate D12	primer working plate 1 D12	10
primer stock plate E1	primer working plate 1 E1	10
primer stock plate E2	primer working plate 1 E2	10
primer stock plate E3	primer working plate 1 E3	10
primer stock plate E4	primer working plate 1 E4	10
primer stock plate E5	primer working plate 1 E5	10
primer stock plate E6	primer working plate 1 E6	10
primer stock plate E7	primer working plate 1 E7	10
primer stock plate E8	primer working plate 1 E8	10
primer stock plate E9	primer working plate 1 E9	10
primer stock plate E10	primer working plate 1 E10	10
primer stock plate E11	primer working plate 1 E11	10
primer stock plate E12	primer working plate 1 E12	10
primer stock plate F1	primer working plate 1 F1	10
primer stock plate F2	primer working plate 1 F2	10
primer stock plate F3	primer working plate 1 F3	10
primer stock plate F4	primer working plate 1 F4	10
primer stock plate F5	primer working plate 1 F5	10
primer stock plate F6	primer working plate 1 F6	10
primer stock plate F7	primer working plate 1 F7	10
primer stock plate F8	primer working plate 1 F8	10
primer stock plate F9	primer working plate 1 F9	10
primer stock plate F10	primer working plate 1 F10	10
primer stock plate F11	primer working plate 1 F11	10
primer stock plate F12	primer working plate 1 F12	10
primer stock plate G1	primer working plate 1 G1	10
primer stock plate G2	primer working plate 1 G2	10

Expected result

Each working plate holds 100 µL at 10 µM in 74 wells.

5Label the plates and store them

primer working plate 1: never return to the freezer a thawed working plate is used once and discarded

Label primer stock plate and primer working plate 1 with the date.
Store every plate at -20 °C.
Thaw one working plate for a run, and throw it away once the run is set up.

Expected result

2 labelled plates at -20 °C, 1 of them ready to run from.