Team:EPF-Lausanne/Protocols/Klenow

 

Klenow fragment synthesis

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 -->The final volum at this stage should be 36.4ul Thermal cycling
 * 3.64ul of NEB buffer (choose this one according to the restriction enzymes you will use later. The klenow fragment enzyme works in all NEB buffer)
 * 0.37ul of BSA 100x
 * X ul for each primer according to concentration
 * Y ul of MQ
 * 94ºC for 5min
 * Ramp down the temperature to 5ºC below your annealing temperature. CAUTION : ramp the temperature down at a MAXIMUM of 0.1ºC/s (to avoid secondary structure formation in your primers)
 * Set the temperature @ 37ºC

Step 1' -->The final volume at this stage should be 39ul Thermal cycling Step 2 : Digestion -->volume at this stage should be 40ul
 * Add 1ul of the Klenow fragment enzyme
 * Add 1.6ul of dNTPs (final concentration of 1mM each). Stock solution of dNTPs : 25mM each
 * Incubate 1h30 @ 37ºC
 * Inactivate the enzyme 20min @ 75ºC
 * Ramp down the temperature to 37ºC (!!! 0.1ºC/s !!!)
 * Add 0.5ul of each enzyme

Thermal cycling Step 3 : Dephosphorylation -->Final volume should be 50ul Thermal cycling (*) This amount of enzyme has been calculated using the following. According to NEB : 1ul needed to dephosphorylate 1-5mg of pUC19 (~2690 bp) in 30 min. So, calculate your molar bp ratio compared to pUC19 and multiply by your supposed DNA mass. The result will give you by how much you have to multiply the phosphatase volume (in ul). You can also let the incubation run for longer (like here 2h) and so use less phosphatase (assume the relationship between ul of enzyme and time of incubation is a linear function). CAUTION : if you use more enzyme volume, your final volume will also be bigger. Make sure to adjust your phosphatase buffer accordingly!
 * Incubate 2h @ 37ºC
 * Inactivate the enzymes 20min @ 80ºC
 * Ramp down the temperature to 37ºC (!!! 0.1ºC/s !!!)
 * Add 5ul of Antartic phosphatase buffer (10X)
 * Add 5ul of Antartic phosphatase enzyme (*)
 * Incubate 2h @ 37ºC
 * Inactivate the enzyme 10 min @ 65ºC
 * Ramp down the temperature to 25ºC (!!! 0.1ºC/s !!!)