PCR Protocol - Part 1 — Transcript

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1. 0:00[Music]
2. 0:06before beginning this protocol students
3. 0:08must fill out the worksheet provided on
4. 0:11the last page of this document this will
5. 0:14allow students to calculate the
6. 0:15appropriate volume of reagents to mix in
7. 0:18the creation of a master cocktail the
8. 0:21purpose of a cocktail is to mix all the
9. 0:23common re agents in a multi- sample
10. 0:25procedure to avoid excess pipetting and
11. 0:28potential errors
12. 0:31in this example of protocol 2A four
13. 0:34students are amplifying the actin and 3
14. 0:36Gene from all four of their DNA samples
15. 0:39this information is filled out on the
16. 0:41left hand side of the
17. 0:44[Music]
18. 0:48worksheet on the right hand side
19. 0:50students use the equations to determine
20. 0:52the number of tubes needed and the
21. 0:55multiplier for reagent
22. 0:57volumes in addition to the 4 d samples a
23. 1:01negative control sample is always
24. 1:03included therefore five tubes are needed
25. 1:06for the PCR
26. 1:08reaction in the creation of the cocktail
27. 1:11pipetting error is accounted for by
28. 1:13adding another sample therefore the
29. 1:15multiplier for this group is
30. 1:17[Music]
31. 1:23six each row of table one represents a
32. 1:26common reagent used in all the samples
33. 1:28undergoing a PCU our
34. 1:31reaction the multiplier is filled out in
35. 1:34all rows of the multiplier column of
36. 1:37table
37. 1:37[Music]
38. 1:47one moving across each row the volume
39. 1:50indicated per reaction is multiplied by
40. 1:53the
41. 1:58multiplier 12 .5 * 6 = 75 microl of red
42. 2:04Tac added to the
43. 2:07cocktail 11.5 * 6 equal 69 microl of
44. 2:12water added to the
45. 2:16cocktail 0.5 * 6 equal 3 microl of each
46. 2:20of the forward and reverse primer added
47. 2:23to the
48. 2:25cocktail finally the sum of these
49. 2:27volumes should be equal to the product
50. 2:29of the fin fin row 75 + 69 + 3 + 3 equal
51. 2:35150 micr of total volume in the
52. 2:40[Music]
53. 2:42cocktail this amount matches 25 *
54. 2:46[Music]
55. 2:556 table two is used as a guide for
56. 2:58labeling of the piece PCR samples as
57. 3:01indicated by the equations this group
58. 3:03will be using five tubes the class must
59. 3:06collectively number the PCR samples to
60. 3:08not confuse samples amongst the
61. 3:13groups in this example these students
62. 3:15have been aside samples numbered 5
63. 3:18through
64. 3:209 the sample names are filled out in the
65. 3:23second column and the primers used for
66. 3:25each sample are filled out in the last
67. 3:27two columns
68. 3:29[Music]
69. 3:44in this example the forward primer is
70. 3:46Act 10 and 3F and the reverse primer is
71. 3:49Act 10 and 3
72. 3:51[Music]
73. 3:58R this same primers are used for all
74. 4:02[Music]
75. 4:11samples the common reagents provided are
76. 4:14molecular biology
77. 4:16[Music]
78. 4:20water a forward
79. 4:22primer which you should be sure to check
80. 4:25for the marking of PCR to ensure that
81. 4:27you are using the appropriate
82. 4:29concentration
83. 4:30[Music]
84. 4:35a reverse
85. 4:43primer and red tack Ready Mix the red
86. 4:46tack will need to be thawed prior to use
87. 4:49but once thawed keep the solution on Ice
88. 4:51to prevent enzyme degradation
89. 4:56[Music]
90. 5:04next prepare the cocktail by using the
91. 5:06volumes and reagents indicated on the
92. 5:08worksheet obtain a new tube and label it
93. 5:11as the
94. 5:13[Music]
95. 5:20cocktail when creating a cocktail it is
96. 5:23best to start with the largest volumes
97. 5:26though red Tac is the largest volume the
98. 5:28solution should be kept on ice as long
99. 5:30as possible so instead the water is
100. 5:33added
101. 5:34first when adding each of the reagents
102. 5:37be sure to use the appropriate micro
103. 5:39piped for the volume being added
104. 5:48[Music]
105. 6:15next the primers are added
106. 6:18[Music]
107. 6:38it is best to pipet small volumes
108. 6:40directly into larger volumes to avoid
109. 6:43loss of the reagent
110. 6:47[Music]
111. 7:15finally add the red Tack and immediately
112. 7:18return the stock to ice
113. 7:31[Music]
114. 7:58mix the solution and centrifuge all
115. 8:00cocktail tubes created by all groups in
116. 8:03the class together
117. 8:05[Music]
118. 8:47press and hold the short button to do a
119. 8:50brief spin
120. 8:59the
121. 9:06[Music]