Team:Warsaw/Calendar-Main/21 April 2009
From 2009.igem.org
Cloning of hly gene into pKSII+ vector
Kamil
Tasks:
- Amplification of hly
- Measurement of concentration of isolates from Yersinia and Listeria
Methods:
-
PCR mixture's composition:
2ul pfu buffer (Fermentas) 1ul MgSO4 (Fermentas) 0,5ul primers 1,5ul dNTPs (10 mM, 01,ul pfu polymerase (Fermentas) 1ul template DNA from Listeria
Solution was topped up with H2O to 20ul.
- PCR program:
hly
300s 95°C
(30s 95°C, 35s 42°C, 150s 72°C)x2
(30s 95°C, 35s 47°C, 150s 72°C)x28
600s 72°C
~ 4°C
- Electrophoretic separation on 1% agarose gel
- Measurement of concentration of isolates on NanoDrop
Results:
- Gel (from left)
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- hly
- hly control -
- Measurement from NanoDrop: + Yersinia - 29,6 ng/ul + Listeria - 50,7 ng/ul
Conclusions:
- 200ng of DNA should be used on sample
- Time of anealing should be prolonged to 45s
- Concentration of Mg should be increased
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