Team:Heidelberg/n4tr1um

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(New page: == Transformation of Bacteria == For enrichment of vectors, E .coli DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 400 ng DNA solutio...)

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~~== Transformation of Bacteria ==~~

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For enrichment of vectors, E .coli DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 400 ng DNA solution added (depending on the concentration of the DNA solution). After a 30-60 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 3 minute incubation on ice. The samples were than rescued by adding 500µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shaking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.

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~~== Isolation of plasmid DNA by alkaline lysis (mini and maxiprep) ==~~

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For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions. For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg. For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.

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~~== Site-directed mutagenesis ==~~

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For removal of unwanted Restriction Sites, a PCR-based site direct mutagenesis protocol was adapted from [http://www.stratagene.com/manuals/200518.pdf Stratgene]. Oligos were designed to have a high (>78°C) Tm ny applying the formula

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~~<math>T_{m} = 81{,}5 + 0{,}41 * (%GC) - \frac{675}N - %mismatch</math>~~

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-== Transformation of Bacteria ==
-For enrichment of vectors, E .coli DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 400 ng DNA solution added (depending on the concentration of the DNA solution). After a 30-60 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 3 minute incubation on ice. The samples were than rescued by adding 500µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shaking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.
-== Isolation of plasmid DNA by alkaline lysis (mini and maxiprep) ==
-For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions. For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg. For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.
-== Site-directed mutagenesis ==
-For removal of unwanted Restriction Sites, a PCR-based site direct mutagenesis protocol was adapted from [http://www.stratagene.com/manuals/200518.pdf Stratgene]. Oligos were designed to have a high (>78°C) Tm ny applying the formula
-<math>T_{m} = 81{,}5 + 0{,}41 * (%GC) - \frac{675}N - %mismatch</math>

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Latest revision as of 12:26, 24 June 2009