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Team:EPF-Lausanne/Protocols/Klenow
From 2009.igem.org
This protocol has been designed in order to get ready-to-ligate small DNA fragments from scratch. The procedure doesn't require any purification step, in order not to loose DNA material due to the small sizes of the fragments. Therefore, it includes a dephosphorylation step, which is crucial for the maximization of the ligation that follows. The all expermiment is performed in a single tube with successive additions of new reactants.
The amount of DNA at the beginning of the reaction is calculated in order to fit the desired amount required for the ligation step that follows the end of the klenow fragment synthesis.
- Design your DNA oligos
- Fix your target DNA concentration that you want at the end (in ng/ul)
- Multiply by 50 to get the total mass of DNA that you want in your tube at the end (the protocol is designed to have a final volume of 50ul)
- Out of the M.W. of your DNA fragment, calculate the number of mol that you would expect at the end --> it gives you the the nb of mol that you will need for each DNA oligos (each oligo must have the same nb mol as for the DNA fragment)
- Out of the concentration of your oligos, you can calculate the volume of each required
Step 1 : Klenow fragment synthesis
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
