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Team:Warsaw/Calendar-Main/1 July 2009
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| - | <h3> | + | <h3>Making Llo fusion with secretion signal peptide</h3> |
<h4>Kuba</h4> | <h4>Kuba</h4> | ||
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<p>Methods:</p> | <p>Methods:</p> | ||
| - | <ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas) | + | <ul><li><p>PCR mixture's composition:</p> |
| + | <pre>2,5ul pfu buffer (Fermentas) | ||
| + | 2,5ul MgSO4 (Fermentas) | ||
| + | 1,5ul primers | ||
| + | 1,5ul dNTPs (10 mM) | ||
| + | 0,5ul pfu turbo polymerase | ||
| + | 1ul template DNA from Listeria, solution was topped up with H2O to 25ul</pre> | ||
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<h3>Quality check (cro PCR product and pKS isolate)</h3> | <h3>Quality check (cro PCR product and pKS isolate)</h3> | ||
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<br> | <br> | ||
<h4>Kuba</h4> | <h4>Kuba</h4> | ||
<ul> | <ul> | ||
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Latest revision as of 18:03, 17 September 2009
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Making Llo fusion with secretion signal peptide
Kuba
Tasks:
- Amplification of llo(hly) for fusion with the secretion signal peptide
Methods:
PCR mixture's composition:
2,5ul pfu buffer (Fermentas) 2,5ul MgSO4 (Fermentas) 1,5ul primers 1,5ul dNTPs (10 mM) 0,5ul pfu turbo polymerase 1ul template DNA from Listeria, solution was topped up with H2O to 25ul
- PCR programs:
hly
4min 95°C
(30s 95°C, 40s 40-48°C, 1min30s 72°C)x3
(30s 95°C, 40s 44-55°C, 1min30s 72°C)x28
10min 72°C
~ 7°C
- Electrophoretic separation on 1% agarose gel
Results:
- Gel (from left)
- M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- 1-5 - samples (annealing temperature increases to from the left to the right)
many unspecific products were obtained (product of the correct lenght marked with an arrow)
Quality check (cro PCR product and pKS isolate)
Kuba
%2Bpks(test).JPG)
- Gel (from the left)
- CRO
- pKS isolate
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
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