Team:Calgary/16 July 2009

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University of Calgary

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JULY 16, 2009

CAROL

Diluting Primers and Primer Extension Polymerase Chain Reaction (PCR)

To create the synthetic sigma 70 promoter library, we have synthesized 32 different primers (16 different forward primers and 16 different reverse primers) to hopefully form 256 different promoters.
Need to dilute primers to 80μM. The following is the concentration of the original primers that were synthesized once water was added to the primers.

Primer	260/280	260/230	Concentration [ng/μL]	Reading/2.5 [ng/μL]	Stock Concentration [μM]	Volume stock into working [μL]	Volume water into working [μL]
sig70-F1	1.98	1.73	20816	832.64	60.07	6.7	43.3
sig70-F2	1.55	1.80	44284	17713.6	1277.91	3.1	46.9
sig70-F3	1.62	1.86	41323	16529.2	1192.46	3.4	46.6
sig70-F4	1.70	1.99	32365	12946	933.96	4.3	45.7
sig70-F5	1.72	2.07	33688	13475.2	972.14	4.1	45.9
sig70-F6	2.07	1.95	45412	18164.8	1310.46	3.1	46.9
sig70-F7	1.64	1.95	39046	15618.4	1126.76	3.6	46.4
sig70-F8	1.43	1.63	45745	18298	1320.07	3.0	47.0
sig70-F9	1.75	2.16	23628	9451.2	681.84	5.9	44.1
sig70-F10	1.72	2.01	32682	13072.8	943.11	4.2	45.8
sig70-F11	1.71	2.07	31494	12597.6	908.83	4.4	45.6
sig70-F12	1.59	1.82	41202	16480.8	1188.97	3.4	46.6
sig70-F13	1.73	2.03	29224	11689.6	843.3	4.7	45.3
sig70-F14	1.41	1.58	45645	18258	1317.2	3.0	47.0
sig70-F15	1.59	1.85	40681	16272.4	1173.9	3.4	46.6
sig70-F16	1.57	1.82	42241	16896.4	1219.0	3.3	46.7
sig70-R1	2.06	2.17	7516	3006.4	216.9	18.4	31.6
sig70-R2	2.04	2.25	15255	6102.0	440.2	9.1	40.9
sig70-R3	1.76	1.85	42503	17001.2	1226.5	3.3	46.7
sig70-R4	2.01	2.21	24012	9604.8	692.9	5.8	44.2
sig70-R5	2.10	2.27	18530	7412	534.7	7.5	42.5
sig70-R6	1.98	2.19	28452	11380.8	821.0	4.9	45.1
sig70-R7	1.94	2.17	32976	13190.4	951.6	4.2	45.8
sig70-R8	2.00	2.20	25379	10151.6	732.4	5.5	44.5
sig70-R9	2.00	2.24	24141	9656.4	696.6	5.9	44.1
sig70-R10	1.94	2.17	30237	12094.8	872.6	4.6	45.4
sig70-R11	1.92	2.16	31497	12598.8	908.9	4.4	45.6
sig70-R12	1.92	2.17	30045	12018	867.0	4.6	45.4
sig70-R13	2.01	2.18	18726	7490.4	540.4	7.4	42.6
sig70-R14	1.88	2.03	36111	14444.4	1042.1	3.8	46.2
sig70-R15	1.98	2.17	10679	4271.6	308.2	13	37
sig70-R16	1.86	2.01	35969	14387.6	1038	3.9	46.1

The sequences for each primer is listed in the following table:

Primer	Sequence
sig70-F1	GCGCGCTCGAGAATAATTCTTAAAATTTATGCTTCCGGCTCG
sig70-F2	GCGCGCTCGAGAATAATTCTTAATATTTATGCTTCCGGCTCG
sig70-F3	GCGCGCTCGAGAATAATTCTTAAGATTTATGCTTCCGGCTCG
sig70-F4	GCGCGCTCGAGAATAATTCTTAACATTTATGCTTCCGGCTCG
sig70-F5	GCGCGCTCGAGAATAATTCTTTAAATTTATGCTTCCGGCTCG
sig70-F6	GCGCGCTCGAGAATAATTCTTTATATTTATGCTTCCGGCTCG
sig70-F7	GCGCGCTCGAGAATAATTCTTTAGATTTATGCTTCCGGCTCG
sig70-F8	GCGCGCTCGAGAATAATTCTTTACATTTATGCTTCCGGCTCG
sig70-F9	GCGCGCTCGAGAATAATTCTTGAAATTTATGCTTCCGGCTCG
sig70-F10	GCGCGCTCGAGAATAATTCTTGATATTTATGCTTCCGGCTCG
sig70-F11	GCGCGCTCGAGAATAATTCTTGAGATTTATGCTTCCGGCTCG
sig70-F12	GCGCGCTCGAGAATAATTCTTGACATTTATGCTTCCGGCTCG
sig70-F13	GCGCGCTCGAGAATAATTCTTCAAATTTATGCTTCCGGCTCG
sig70-F14	GCGCGCTCGAGAATAATTCTTCATATTTATGCTTCCGGCTCG
sig70-F15	GCGCGCTCGAGAATAATTCTTCAGATTTATGCTTCCGGCTCG
sig70-F16	GCGCGCTCGAGAATAATTCTTCACATTTATGCTTCCGGCTCG
sig70-R1	GCGGGATCCAATTGCACGTAAAATACGAGCCGGAAGCATAAA
sig70-R2	GCGGGATCCAATTGCACGTAATATACGAGCCGGAAGCATAAA
sig70-R3	GCGGGATCCAATTGCACGTAACATACGAGCCGGAAGCATAAA
sig70-R4	GCGGGATCCAATTGCACGTAAGATACGAGCCGGAAGCATAAA
sig70-R5	GCGGGATCCAATTGCACGTATAATACGAGCCGGAAGCATAAA
sig70-R6	GCGGGATCCAATTGCACGTATTATACGAGCCGGAAGCATAAA
sig70-R7	GCGGGATCCAATTGCACGTATGATACGAGCCGGAAGCATAAA
sig70-R8	GCGGGATCCAATTGCACGTATCATACGAGCCGGAAGCATAAA
sig70-R9	GCGGGATCCAATTGCACGTAGAATACGAGCCGGAAGCATAAA
sig70-R10	GCGGGATCCAATTGCACGTAGTATACGAGCCGGAAGCATAAA
sig70-R11	GCGGGATCCAATTGCACGTAGGATACGAGCCGGAAGCATAAA
sig70-R12	GCGGGATCCAATTGCACGTAGCATACGAGCCGGAAGCATAAA
sig70-R13	GCGGGATCCAATTGCACGTACAATACGAGCCGGAAGCATAAA
sig70-R14	GCGGGATCCAATTGCACGTACTATACGAGCCGGAAGCATAAA
sig70-R15	GCGGGATCCAATTGCACGTACGATACGAGCCGGAAGCATAAA
sig70-R16	GCGGGATCCAATTGCACGTACCATACGAGCCGGAAGCATAAA

Stock dATP, dGTP, dCTP, dTTP from Invitrogen was diluted from 100mM to 0.5mM.
See how the primer extension PCR is done in the protocol page.
Both the varying promoters and the vector, pCS26, was digested using enzymes, XhoI and BamHI overnight. The vector was incubated at 37^oC and the promoter was digested for 2 hours at 16^oC and then overnight at room temperature (~21^oC).

CHINMOYEE