PacBioPrimerPCROptimization

	A	B	C	D	E	F	G	H	I	J	K	L	M	N
1	Primers								Template
2	Primer Name	Oligo ID	Tm (C)	Concentration (mM)					Sample	Name	Concentration (ng / ul)	Volume (ul)	Total DNA mass (ng)	Yeild
3	PacBioBarFwd-0	oEH30	58	100		Primer concentration	0.5		1	T7InitMix+TXN	11	16	176	0.44
4	PacBioRev-0	oEH60	58	100		Reaction volume	20		2	T7INITMIX-TXN	29.7	10	297	0.7425
5						Template Mass / Rxn	10
6	Primer Dilutions
7	Primer Name	Dilution target concentration (mM)	Dilution total volume (ul)	Primer volume (ul)	npH20 volume (ul)
8	PacBioBarFwd-0	5	20	1	19
9	PacBioRev-0	5	20	1	19
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14	Sample	Total volume (ul)	5x Phusion GC buffer	10 mM dNTPs	Diluted PacBioBarFwd-0	Diluted PacBioRev-0	Template	Template volume	PhusionU volume
15	1	20	4	s	2	2	T7InitMix+TXN	0.909	0.2
16	2	20	4	s	2	2	T7InitMix+TXN	0.909	0.2
17	3	20	4	s	2	2	T7InitMix+TXN	0.909	0.2
18	4	20	4	s	2	2	T7InitMix+TXN	0.909	0.2
19	5	20	4	s	2	2	T7INITMIX-TXN	0.337	0.2
20	6	20	4	s	2	2	T7INITMIX-TXN	0.337	0.2
21	7	20	4	s	2	2	T7INITMIX-TXN	0.337	0.2
22	8	20	4	s	2	2	T7INITMIX-TXN	0.337	0.2
23	9	20	4	0.4	2	2	NA	0	0.2
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26								MM Multiplier	1.2
27	Master mixes
28	Mix Name	Template	npH20	5x Phusion GC buffer	10 mM dNTPs	Diluted PacBioBarFwd-0	Diluted PacBioRev-0	Total Volume
29	No Template or Pol	0	129.12	43.2	0.48	21.6	21.6	216
30
31		No Template or Pol (ul)	PhusionU
32	Pol +	177.84	2.16
33
34		Pol + (ul)	Volume template
35	Pol + T7InitMix+TXN	76.364	3.636
36
37	Pol + T7INITMIX-TXN	78.652	1.348
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40	Reactions
41	Sample number	Master Mix	Master Mix volume	DNA Mass (ng)	Annealing temp (C)	Annealing time (s)	Extension time (s)	Cycles
42	1	Pol + T7InitMix+TXN	20	9.999	45	30	66	35
43	2	Pol + T7InitMix+TXN	20	9.999	48.4	30	66	35
44	3	Pol + T7InitMix+TXN	20	9.999	54.4	30	66	35
45	4	Pol + T7InitMix+TXN	20	9.999	58.9	30	66	35
46	5	Pol + T7INITMIX-TXN	20	10.0089	45	30	66	35
47	6	Pol + T7INITMIX-TXN	20	10.0089	48.4	30	66	35
48	7	Pol + T7INITMIX-TXN	20	10.0089	54.4	30	66	35
49	8	Pol + T7INITMIX-TXN	20	10.0089	58.9	30	66	35
50	9	Pol +	20	0	45	30	66	35
51	10	No Template or Pol	20	0	48.4	30	66	35
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