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From 2009.igem.org

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Revision as of 19:04, 20 October 2009

Klenow protocol New protocol used with no purification step, everything is prepared to go trough all reaction up to the end of the digestion, ready for the ligation without going through any step of purification. This will avoid losing DNA due to the small size of the TrpO (127bp). Goal: we want 100ng/ul od DNA at the end length of TrpO:127bp molecular weight: 78,522 . 10^3 g/mol → 100ng/ul in 50ul → 6,367.10^-11 mol of TrpO a the end, that's to say 6,367.10^-11 of each primer Trp Operon-Rev: 1,560.10^3M → 2.56 ul Trp Operon-Fwd: 1,560.10^3M → 2.56 ul first make a dilution at 25ul in 500ul → 8ul in 492ul of MQ

1. Klenow dNTPs final concentr = 1mM 3.64ul of NEB2 0.37ul of BSA 100x 2.56ul of each primer at 25uM 32.38ul of MQ → final: 36.4ul

Thermal cycler: 94°c for 5min 0.1°C/s to 74°C for 5 min 0.1°C/s to 37°C Annealing temperature: 79.1°C (due to some website)

1.' 1ul Klenow + 1.6ul dNTPs = vol 39ul Incubate 1h30 at 37°C, then inactivate 20 min at 75°C : 0.1°C/s to 37°C

2. Digestion Add 0.5 ul of each enzyme (EcoRI - HF / NheI) → vol: 40.0ul Incubate 2h at 37°C, then inactivate 20min at 80°C. Then cool down at 37°C (0.1°C/s)

3. Dephosphorylation Add 5ul of antartic phosphatase buffer + 5ul of phosphatase enzyme → total volume: 50ul Incubate 2h at 37°C, then inactivate 10min at 65°C. Then cool down slowly (0.1°C/s) 1ul is needed to dephosphorylate 1-5mg of vector pUC19

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