User:Ana R. Pengelly/Notebook/His/2010/11/29

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Transformation of the ccdB bacteria with the pDESTR3R4 Φ31 attB vector
1.vials of the ccdB survival strain competent cells were thawed on ice(for 10 min) 2.1-5 μL of DNA was added and mixed by tapping the tubes. 3.Cells were incubated on ice for 30 min 4.Cells were heat shocked for 42 sec at 42 °C 5.Vials were incubated on ice for 2 min 6.300 μL were added to each vial 7.Vials were incubated at 30°C for 1h30min with shaking at 220 rpm 8.Cells were spun down at 6000 rpm for 3 min and resuspended in 200 or 100 μL of SOC 9.100 to 150 μL were plated and incubated 24 hrs at 30 °C ( you can look at then after 16 hrs) Samples -pDONR221 was used as a positive control (0.5μL) -pDESTR3R4 Φ31 attB was transformed using either 1 or 2μL (these 2 transformations were divided in 2 equal volumes, and one was incubted at 30°C on Cm+Amp plates or at 37°C on Cm+Amp plates or at 30°C on Amp plates. To evaluate the influence of temperature and Cm. -Negative control without any DNA.
 * transformation procedure


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