Team:Heidelberg/Tyrocidine week18 biobrickmod

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Contents

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