Question

biochemistry question

Nucleic Acids 33 boslari, no NO E Let's Make a PCR Reaction Reaction Mixture: You have stock DNA of 100 ng/uL that you wish to amplify. You have stocks of 10X PCR Buffer, 10 uM of the forward and reverse primers, 25 mM MgCl2, 50 mM dNTPs , and Tag that is 10 Units/UL. Create the Recipe for a 20 PL total PCR that uses 50 ng of DNA, 2.5 mM primers, 5 mM MgCl2, 5 mM dNTPs, and 2.5 units of Taq. Use water to make up any remaining volume.

Answer 1

PCR recipe.
PCR total volume = 20 ul
PCR buffer stock = 10X
so use 2 ul of stock to get 1 x for 20 ul total volume.

Target DNA stock is 100 ng/ul. We need to add only 50 ng. So only 0.5 ul of stock target DNA is needed.
Logic: 100 ng = 1 ul; 50 ul = ? , cross multiply = 50*1/100 = 0.5ul]

Primers = 10 mM stock of forward and reverse
We need 2.5 mM, so use 4 ul of primers. {2.5/10}*20 ul =5 ul

MgCl2 = stock is 25 mM, we need 5 mM, so use 5/25*20 = 4 ul

dNTPs, stock = 50 mM, we need 5 mM, so use 5/50*20 = 2 ul

Taq polymerase, stock is 10 units/uL, we need 2.5 units. So use 2.5/10 = 0.4 ul of stock taq.

• PCR buffer = 2 $\mu$L
• Target DNA = 0.5 $\mu$L
• Primers =5 $\mu$L
• MgCl2 = 4 $\mu$L
• dNTPs = 2 $\mu$L
• Taq polymerase= 0.4 $\mu$L
• total = 13.9 $\mu$L
• Water = 20-13.9 = 6.1 $\mu$L
• Grant total = 20 $\mu$L