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EPF-Lausanne/7 August 2008
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| + | ===MiniPrep=== | ||
| + | Redid miniprep of E1010, F1610, I1466, R0040. | ||
| + | All were 60ng and more. | ||
| + | |||
| + | ==Ligations== | ||
| + | Doing ligation of E1010-F1610 and R0040 and R0034 | ||
| + | Ligation protocol : | ||
| + | Following the protocol from OpenWetWare, but with our T4 ligase the ligation time is 4 hrs at 15 degrees. We perform the ligation with diluted solutions of vector as well as undiluted, because the vector is in much higher concentration than the insert. Proportion of insert to vector is 6:1 molar, which means 1:1 mass, because insert is 700 bp long and vector about 4000, so the ratio is already 6:1 if we add the same mass of both. Two controls are made, with just the diluted vector present in the ligation reaction. | ||
| + | |||
| + | ==Transformations== | ||
| + | We use the ligation reaction to transform our Top10 competent cells according to the igem protocol. We have Amp plates and the resistance of the vector is to Kan and Amp. We plate Kan on an Amp plate. We also make new plates of Kan and Amp. | ||
Latest revision as of 12:11, 7 August 2008
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MiniPrep
Redid miniprep of E1010, F1610, I1466, R0040. All were 60ng and more.
Ligations
Doing ligation of E1010-F1610 and R0040 and R0034 Ligation protocol : Following the protocol from OpenWetWare, but with our T4 ligase the ligation time is 4 hrs at 15 degrees. We perform the ligation with diluted solutions of vector as well as undiluted, because the vector is in much higher concentration than the insert. Proportion of insert to vector is 6:1 molar, which means 1:1 mass, because insert is 700 bp long and vector about 4000, so the ratio is already 6:1 if we add the same mass of both. Two controls are made, with just the diluted vector present in the ligation reaction.
Transformations
We use the ligation reaction to transform our Top10 competent cells according to the igem protocol. We have Amp plates and the resistance of the vector is to Kan and Amp. We plate Kan on an Amp plate. We also make new plates of Kan and Amp.
