PCR Method Video — Transcript

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1. 0:09PAUL SCHWEIN: Hello, my name is Paul Schwein.
2. 0:11I'm a graduate student here at Harvard University,
3. 0:13and I'm going to tell you how to run a PCR.
4. 0:16PCR, or polymerase chain reaction,
5. 0:18is a standard laboratory technique
6. 0:20used to amplify a specific sequence of DNA many times.
7. 0:24PCR has a number of applications,
8. 0:26including forensics, DNA sequencing,
9. 0:28and diagnosing genetic diseases.
10. 0:32Before starting PCR, I'll need to design primers
11. 0:35to target the region within the human genome
12. 0:38that I want to amplify.
13. 0:39Primers are short sequences of DNA
14. 0:42that bind to specific regions within the genome
15. 0:44and tell DNA polymerase where to initiate replication.
16. 0:48So here, I have a fragment of the human genome,
17. 0:51and in the center is the region that I want to amplify.
18. 0:54In order to amplify this region, I'll
19. 0:56need to design both a forward primer and a reverse primer.
20. 1:00My forward primer will in anneal here,
21. 1:07and DNA polymerase will amplify in the three prime direction.
22. 1:13Then I'll design a reverse primer that anneals here,
23. 1:23and again, DNA polymerase will amplify
24. 1:25in the three prime direction.
25. 1:30After many cycles of PCR, the most predominant DNA product
26. 1:34that will be present in the solution
27. 1:35will look something like this.
28. 1:38The sequence will exactly match my amplified region,
29. 1:45and the sequence here will be identical to my forward primer.
30. 1:50And the sequence here will be identical to my reverse primer.
31. 1:56So now it's time to set up the PCR.
32. 1:58I'll be adding the components from largest volume
33. 2:00to smallest volume.
34. 2:02First is water.
35. 2:10I'll be adding these components to the PCR tube, which
36. 2:14is very small and has thin plastic lining
37. 2:17to enable it to change temperature quickly.
38. 2:20The reactions also happen in small volumes,
39. 2:22so that they can change temperature very quickly.
40. 2:29Next, I'll be adding the PCR buffer.
41. 2:33This buffer contains Tris-HCl and KCl.
42. 2:38And it's meant to stabilize the pH between 8.3 and nine.
43. 2:47The buffer is at a 10x concentration,
44. 2:51and its final volume within the reaction should be 1x.
45. 2:55So I'm adding two microliters to my 20 liter reaction.
46. 3:06Next, I'll be adding the DNTPs.
47. 3:09These are the building blocks of DNA,
48. 3:11and this is what DNA polymerase is
49. 3:14going to be synthesizing together
50. 3:16as it creates the new strands.
51. 3:26Next, I'll add magnesium chloride.
52. 3:28This is important for the reaction.
53. 3:30It's an important stabilizer for the DNA backbone
54. 3:33and stabilizes its interaction with DNA polymerase.
55. 3:51Next, I'll be adding my template DNA.
56. 3:54I'm just going to add one nanogram of template DNA.
57. 4:13Next, I'll need to add my primers.
58. 4:15I'll be adding the forward primer and the reverse primary
59. 4:17separately.
60. 4:18The final concentrations of these primers
61. 4:20will be one micromolar, but you may
62. 4:23need to troubleshoot this for a different PCR.
63. 4:26You may need more or less.
64. 4:28So first is the forward primer, and next, the reverse primer.
65. 4:54And finally, I'll add DNA polymerase,
66. 4:57which will carry out the DNA synthesis reaction.
67. 5:10There we go.
68. 5:11And once I've mixed and spun down my sample,
69. 5:12I'll be ready to go.
70. 5:15I just finished showing you all of the components that
71. 5:18go into one PCR reaction.
72. 5:20Oftentimes, all of the components
73. 5:22of PCR except for the template and primers that you bring in
74. 5:25are pre-assembled into what's called a master mix.
75. 5:28Master mixes make pipetting more accurate, and convenient,
76. 5:31and should be used when setting up multiple PCR reactions.
77. 5:35I used a master mix to setup these three PCR reactions.
78. 5:40Before I set up the PCR program, I
79. 5:42went to quickly review the three steps of a PCR cycle.
80. 5:46In the first step, double stranded genomic DNA
81. 5:49is heated up and separated into two single strands.
82. 5:53This step is called denaturation,
83. 5:57and it occurs at 95 degrees Celsius.
84. 6:01Once we have two single strands, we
85. 6:03lower the temperature to allow for the primers we
86. 6:05designed to bind to the complementary regions
87. 6:08within the genome.
88. 6:09This step is called annealing.
89. 6:14And in our PCR, this is happening
90. 6:15at 68 degrees Celsius.
91. 6:17But this may differ based on the melting temperatures
92. 6:20of the primers you design.
93. 6:22Then in the last step, DNA polymerase
94. 6:24moves in and begins replication, generating two
95. 6:28double stranded pieces of DNA.
96. 6:30This step is called extension, and it
97. 6:34happens at 72 degrees Celsius.
98. 6:38Now it's time to set up the program.
99. 6:41Next, I'll need to write a PCR program into the Thermo Cycler
100. 6:44before adding my samples.
101. 6:47Here's the Thermo Cycler screen that
102. 6:49shows a schematic of the PCR reaction
103. 6:51with each step from left to right,
104. 6:54and steps two through four here will be
105. 6:56repeated within each PCR cycle.
106. 7:00So the first step is the initial denaturation step.
107. 7:02I want this to be at 95 Celsius, and I want
108. 7:04this to last for five minutes.
109. 7:07Next is the denaturation step within each cycle.
110. 7:11I want this at 95 Celsius, again, for 30 seconds.
111. 7:16Next is the annealing step.
112. 7:18I'll calculate the temperature for this
113. 7:19using the average of the TMs of each of my primers,
114. 7:22so this will be at 68 Celsius for this primer pair,
115. 7:26again, for 30 seconds.
116. 7:29And then the last step of the cycle is extension.
117. 7:31I want this at 72 Celsius for 30 seconds.
118. 7:37Then this cycle will repeat 34 times.
119. 7:40When the cycle is over, I'll do a final extension step,
120. 7:44so that DNA polymerase has an opportunity
121. 7:46to finish up any copies that weren't completed.
122. 7:49So I want this at 72 Celsius for seven minutes.
123. 7:55And then finally, I want the Thermo Cycler
124. 7:57to stay at 12 degrees forever, until I can come to the machine
125. 8:00and get my samples.
126. 8:01So they stay nice and cool.
127. 8:04Now I'm ready to add my samples to the Thermo Cycler.
128. 8:09I'll place them here in the center.
129. 8:19I'll close the lid and tighten it.
130. 8:22You may not need to tighten the lid on your PCR machine,
131. 8:29and I'll click Run.
132. 8:31I'll come back when my PCR samples are ready.
133. 8:35My PCR program is now complete.
134. 8:37I expect that my target DNA sequence
135. 8:39got amplified many times.
136. 8:41One of the most common ways of analyzing your PCR
137. 8:44is to run a DNA gel.
138. 8:46You've now seen me assemble and run
139. 8:47a polymerase chain reaction.
140. 8:49I hope this helps you in your laboratory work.
141. 8:51Thank you for watching.