20NOV17 In vitro protein synthesis of the IRM6-13

20NOV17

I estimated the DNA concentration in the starting material I have. I used the Qubit for this estimation. I need to know the DNA concentration because I need to have 250ng DNA per 25μl of the  reaction (according to the manufacturer's instructions). This means that I need to have  a final concentration of

250ng    in     25μl
   x         in        1μl 


x = 250ngμl/25μl
x = 10ng/μl

So if we measure the initial DNA concentration and we know that the final reaction volume is 25 μl then its easy to figure out how much template DNA I need to add.

The concentration of the DNA template was calculated with Qubit as follows in ng/μl:
IRM6 : 54.8
IRM7 : 30
IRM8 : 39.4
IRM9 : 39
IRM10 : 21
IRM11 : 50.8
IRM12 : 57.4
IRM13 : 48

I followed the instructions in the invitro protein synthesis kit which says to add the solution A, then the solution B, then the required water (so that the volume of the final reaction will be 25μl) and the DNA template (so that we have 10ng/μl always).  The volumes added were as follows:

I incubated the reaction at 37oC for 4 hours. I freeze then produce protein at -20oC.