How to do PCR

Green Promega Master (Monster) Mix

Do it yourself from invidual components

Green Promega Master (Monster) Mix

Common Temperature Cycle (30 seconds per kb of DNA amplified):

• Step 1: 95° C 5 minutes (denature template)
• Step 2: 95° C 30 seconds (denature dsDNA)
• Step 3: 55° C 30 seconds (T_m minus 5 degrees)
• Step 4: 72° C 30 seconds (amplify about 1 minute for each 1000 bp)
• Step 5: Repeat Steps 2 through 4, 29 more times
• Step 6: Store at RT°

REAGENT*	VOLUME (µL)	FINAL CONC.
water	50 - (X + Y + Z) µL	N/A
2X Master Mix (with buffer, Taq, dNTPs, MgCl2**)	50 µL	1X
template DNA (~1 ng DNA)	X ( < 1 µL)	1 ng
primer #1	Y µL	1 μM
primer #2	Z ML	1 μM
FINAL VOLUME	100 µL

*All reagents should be thawed quickly and then stored on ice.
**Click here to read about the importance of magnesium concentration!!

Do it yourself from invidual components

In order to do PCR(polymerase chain reaction), you need the following components:

• template DNA
• reaction buffer with the appropriate amount of MgCl₂
• dNTPs
• forward primer
• reverse primer
• Taq DNA polymerase

The question is, how much of each? Traditionally, PCR is done in 100 μl volumes and the reaction buffer comes as a 10X stock solution (meaning it is 10 times too concentrated and must be diluted). For our labs, the dNTPs are in a 20X stock solution; the primers and template are in 100X stock solutions; and the Taq is in a 200X stock solution. Therefore, you should set up a table, fill in the blanks, and add the reagents in the order presented below (always protect your enzymes by adding them last - they are touchy creatures and expensive).
Once you have your mixture ready, put it in the thermal cycler and start the process. We will be using the program called IDH with the heated lid ENABLED.

IDH Temperature Cycle:

• Step 1: 95° C 5 minutes (denature template)
• Step 2: 95° C 1 minute (denature dsDNA)
• Step 3: 55° C 1 minute (T_m minus 5 degrees)
• Step 4: 72° C 1 minute (amplify about 1 kb per minute)
• Step 5: Repeat Steps 2 through 4, 29 more times
• Step 6: Store at RT

REAGENT*	VOLUME (μl)	FINAL CONC.
water		N/A
10x buffer with magnesium**		1X
or buffer w/o Mg		1X
plus 25 mM MgCl2 stock		varies (often 1.5 mM)
template DNA (~1 ng gDNA)		1 ng
dNTPs (4 mM stock for each base)		(200μM stock for each base)
primer #1		1 μM
primer #2		1 μM
Taq* (500 units/1 μL stock)		2.5 units
FINAL VOLUME	100

*The Taq must be kept in the special "cold block" in order to keep the enzyme as cold as possible while it is being pipetted. All other reagents should be thawed quickly and then stored on ice.

**Click here to read about the importance of magnesium concentration!!

Lab Schedule Outlined

Lab Schedule In Context of Research Project