Now that you have planned out what fragments you need, see that some are unique

Question

Now that you have planned out what fragments you need, see that some are unique and some are repeats. Plan how you are going to set up each PCR reaction 50 ul total volume, using 2XQ5 master mix (this contains the buffer, dNTPs and polymerase). We recommend making two master master mixes. How will you do this? Think about reagents that are common and reagents that are different. What reagents can you combine in your master mixes and which do you have to add separately'? What PCR run parameters do you propose programming the PCR machine with? Refer to the NEB 2X Q5 protocol for suggested parameters

Explanation / Answer

Ans- If we want to make two master mixes, it can be simply performed by mixing the dNTPs, buffer, Taq polymerase and MgCl2 . The reagents which will be common are dNTPs, buffer and Taq polymerase. The reagent whose concentration will be different in two master mixes are MgCl2 and nuclease free water and primers. All the reagent can be combine together except the MgCl2, NFW and primers.

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