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	==RFC10 Standardization of Tyc Modules==		==RFC10 Standardization of Tyc Modules==
	===new Primer for B1 and a new fw Primer for TycC5===		===new Primer for B1 and a new fw Primer for TycC5===

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Revision as of 11:30, 4 October 2013

Contents 1 RFC10 Standardization of Tyc Modules 1.1 new Primer for B1 and a new fw Primer for TycC5 1.2 Amplification Tyc B1 and Tyc C5 1.3 Results of Gel extraction Tyc B1 and Tyc C5 1.4 Restriction Digest 1.4.1 Tyc ADeltaCom, B1, C5, C6 (AT_01 to AT02, AT_09 to AT_10, AT_11 to AT_06, AT_07 to AT_08; xx kb; xx kb ; xx kb; xx kb) with EcoRI-HF and PstI 1.4.2 Restriction Digest of pSB1C3 (xx kb) with EcoRI-HF and PstI 1.5 Nucleotide Removal and Concentration Measurement of digested pSB1C3, Tyc ADeltaCom, B1, C5, C6

RFC10 Standardization of Tyc Modules

new Primer for B1 and a new fw Primer for TycC5

Amplification Tyc B1 and Tyc C5

29-08-2013

Reaction I

what	µl
Bacillus	1
AT09/11	2
AT10/06	2
Phusion Flash 2x Master Mix	10
DMSO	1
ddH20	4

Conditions II

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
14	98	0:01
	65 ↓0.5	0:05
	72	0:55
16	98	0:01
	68	0:05
	72	0:55
1	72	5:00
1	10	inf

Results of Gel extraction Tyc B1 and Tyc C5

Sample	concentration ng/µl	Adsorption 260	Adsorption 280
H2O	0.1	0.002	0.015
B1	43.5	0.871	0.461
C5	23.9	0.477	0.296

Restriction Digest

Tyc ADeltaCom, B1, C5, C6 (AT_01 to AT02, AT_09 to AT_10, AT_11 to AT_06, AT_07 to AT_08; xx kb; xx kb ; xx kb; xx kb) with EcoRI-HF and PstI

what	µl
Tyc ADeltaCom, B1, C5, C6	20
EcoRI-HF	1
PstI-HF	1
Buffer CutSmart	5
dd H2O	24

Inkubate 37°C 2h

Restriction Digest of pSB1C3 (xx kb) with EcoRI-HF and PstI

what	µl
pSB1C3	3
EcoRI-HF	0.5
PstI-HF	0.5
Buffer CutSmart	5
dd H2O	41

Inkubate 37°C 2h

Nucleotide Removal and Concentration Measurement of digested pSB1C3, Tyc ADeltaCom, B1, C5, C6

Concentration measurements showed that DNA was lost with Nucleotide Removal.

Reamplification and RFC10_Modules#Restriction_Digestas above