IVT-optimization

	A	B	C	D	E	F	G	H	I	J	K	L	M	N	P	Q
1	Polymerase	Template	Polymerase Concentration (units/ml)	Template concentration (ng/ul)	Template Length kb)	rNTP Stock concentration (mM)	DTT stock concentration (mM)
2	NEB T7	pFC9	50,000	300	3.5	25	100
3
4
5	Template concentration (nM)	Polymerase Concentration (units / ul)
6	129.8701299	50
7
8						Check template concentration calc	Total volume calc		Lots
9	Polymerase Dilutions				Desired	5	20		NEB 10x Transcription buffer	10073286
10	Dilution number	1x Transcription buffer	NEB T7	Concentration (units / ul)	Calculated	5	18.6		NEB T7	10117895		0.00025	250
11	STOCK	0	5	50		TRUE	FALSE		NEB rNTPs	10085968
12			Volume previous dilution						promega DTT	38464
13	1	2.5	2.5	25
14	2	2.5	2.5	12.5		35.95238095
15	3	2.5	2.5	6.25
16	4	2.5	2.5	3.125
17	5	2.5	2.5	1.5625
18	6	2.5	2.5	0.78125
19	Total individual dilution volume	5
20
21
22	Sample number	Total reaction volume (ul)	Template concentration (nM)	Polymerase Dilution	Polymerase Dilution volume (ul)	Units Polymerase	Template volume	Template mass (ng)	Template concentration (ng / ul)	Pol units / ul / nM Template	10x Transcription buffer	rNTPs	DTT	npH20	NanoMoles Template	nanoMoles Template / units Pol
23	CTRL	20	5	CTRL	0	0	0.77	231	11.55	0	2	0.4	1	13.83	0.0001	0
24	STOCK	20	5	STOCK	2	100	0.77	231	11.55	20	2	0.4	1	13.83	0.0001	18
25	1	20	5	1	2	50	0.77	231	11.55	10	2	0.4	1	13.83	0.0001	2
26	2	20	5	2	2	25	0.77	231	11.55	5	2	0.4	1	13.83	0.0001	4
27	3	20	5	3	2	12.5	0.77	231	11.55	2.5	2	0.4	1	13.83	0.0001	8
28	4	20	5	4	2	6.25	0.77	231	11.55	1.25	2	0.4	1	13.83	0.0001	16
29	5	20	5	5	2	3.125	0.77	231	11.55	0.625	2	0.4	1	13.83	0.0001	32
30	6	20	5	6	2	1.5625	0.77	231	11.55	0.3125	2	0.4	1	13.83	0.0001	64
31
32	Total	160					6.16				16	3.2	8	110.64
33
34	Master mixes
35	Multiplier	1.2
36
37	npH20	10x Transcription buffer	Template	rNTPs	DTT	Total Volume
38	132.768	19.2	7.392	3.84	9.6	172.8	21.6
39
40
41	Sample setup
42	Sample Number	Volume Transcription master mix	Polymerase Dilution	Polymerase Dilution Volume	npH20	Pol units / ul / nM Template	Template concentration (nM)	Polymerase concentration (units)
43	CTRL	18	CTRL	0	2	0	5	0
44	STOCK	18	STOCK	2	0	20	5	100
45	1	18	1	2	0	10	5	50
46	2	18	2	2	0	5	5	25
47	3	18	3	2	0	2.5	5	12.5
48	4	18	4	2	0	1.25	5	6.25
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50
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56
57			1				0.05	0.1	0.15	0.2	0.25
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59
60				1.55
61				35.96287703
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100