An example of PCR condition: PCR conditions: 95°C x5 minutes -35 cycles: 94°C x

Question

An example of PCR condition: PCR conditions: 95°C x5 minutes -35 cycles: 94°C x 30 seconds 54°C x 40 seconds 72°C x 60 seconds 72°C x 10 minutes 4°C hold PCR Master Mix for 16S rRNA amplification Primer 27F (20pmol/L) Primer 1492R (20pmol/uL) 1.0 1.0 10.0 0.3 50.0 | ffe Taq Polymerase *5X PCR buffer contains MgCl, KCI, and dNTPs C, V,C You are preparing the DNA template for PCR to amplify the 16S rRNA gene. How would you dilute the total DNA you isolated with a known concentration of 500 ng/ul to the concentration of 10 ng/ul, so it can b used for the Master Mix above? 1. When setting up this PCR, the final volume in the reaction tube is set be 50 . (a) How do you determine the required amount of 5x PCR 10 ? (b) what would be the amount of 5 X PCR buffer in a 301 reaction? (c) Why do you add water? 2.

Explanation / Answer

1. our concentration given is 500ng DNA in 1micro liter and our required conc. is 10ng DNA in 1 microliter

we can use formula of conc.given * volume given/ conc.required= volume required

conc. given is 500ng/microliter

conc. required = 10ng/microliter

volume given is 1 microliter

hence 500*1/10=50

when we add 50 microliter of D/W to our 500ng/microliter sample it will make (500/50) = 10ng/microliter

2.a we are adding 5x buffer in reaction with volume of 10microliter which is a stander procedure of reaction. if we have 10X buffer than we will add 5microliter. and if we have 20X than we add 2.5micro liters.

2.b. if we add 10microliter in 50microliter reaction

than what to add in 30 microliter reaction?

50microliter reaction ---------------10microliter

30microliter reaction --------------?

30*10/50= 6microliter

6microliter 5X PCR buffer in 30 microliter reaction similarly,

2.c. to balance the reaction in all PCR reaction tubes we adds water up to 50 microliter so that all tubes will be in balance volume. all in addition, water will provide more area to molecules like dNTPS, MgCl2 or enzymes to work smoothly.it also dilutes the component concentration and it is nuclease free water hence it is avoid the activity of nuclease and protect the DNA.

Related Questions

Navigate

• Browse (All)
• Browse A
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.