How to Harvest Plasmid DNA with Minipreps — Transcript

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1. 0:30Hi! Today we'll be extracting plasmid DNA from bacteria, it's a protocol is usually called a miniprep
2. 0:37It's called minipreps because we will be extracting DNA from a small
3. 0:41volume of bacterial culture. You can also do midipreps and maxiprep
4. 0:45from bigger volumes, but we won't discuss it today
5. 0:49So to start you have to start a bacterial culture
6. 0:52for this we need a culture tube and some media
7. 1:00To keep it sterile we will be working around the source of heat
8. 1:08I will now pour some media into the cell culture tube
9. 1:21About five mL is enough
10. 1:26We also need to add antibiotics to the medium. In this case it's kanamycin
11. 1:40The antibiotic is needed to prevent other contaminants from growing this way only our plasmid
12. 1:46our bacteria containing our plasmid will grow in this medium
13. 1:53And now we have to pick one colony one single colony from a plate. We also are doing this in sterile environment
14. 2:10So now all we have to do is to label our tube and put it to incubate at 37 degrees overnight
15. 2:21So now our culture has grown and we are ready to prepare the miniprep
16. 2:27actually the best way is to take a 2 milliliter tube and
17. 2:34Extract plasmid DNA from 2 milliliter of bacteria, you don't have to be in sterile environment for this step
18. 2:40so I just poured the 2 milliliters into the tube
19. 2:45And we will now have to centrifuge it around 2 minutes, 2 to 3 minutes at 6000 RPM
20. 2:56to avoid damaging the cells don't centrifuge too fast
21. 3:06I'll actually use another tube of two milliliters to equilibrate the centrifuge
22. 3:29And it will centrifuge the cells to keep all the bacteria on the bottom of the tube in the pellet
23. 3:53So our pellets are ready. They are here in the bottom of the tube, and we're now ready to continue with the mini prep
24. 4:00For all the next steps I will put gloves to protect
25. 4:03DNA contained in the samples from the DNase so that could be on my hands
26. 4:15So now we have to discard the supernatant by just pouring it out, and we only keep the pellet on the bottom
27. 4:23First step is to add cell lysis buffer
28. 4:27Usually for minipreps you'll be using a commercial kit and all the solutions already come prepared for you
29. 4:35The kits differ a little bit between the brands, but the principle is the same.
30. 4:39First we adding the cell resuspension buffer 200 microliters of this buffer
31. 4:46And the point is to resuspend the cells in this solution
32. 4:58So we pour it into the Tube and then
33. 5:02You can either gently
34. 5:05pour the solution or the easier way
35. 5:12Is to do the following, and then the pellet is resuspended
36. 5:22Next step we will add the cell lysis buffer. This will pour
37. 5:28the membranes open, it will disrupt the cell Membrane and
38. 5:33the cell contents will pour inside the solution
39. 5:40so 250 microliters of the lysis buffer
40. 5:49And we have to mix it gently to prevent disrupting the DNA
41. 6:01The next step is to add the neutralization buffer
42. 6:07It will neutralize the ph and would also cause
43. 6:12chromosomal DNA to precipitate and only the
44. 6:16plasmid DNA will arrest in the solution
45. 6:30350 microliters of neutralization buffer and you
46. 6:36will see that the solution becomes
47. 6:38troubled and the precipitate appears
48. 6:43We also mix the tube gently to avoid disrupting the DNA
49. 6:50Now we will have to separate the
50. 6:55separate the precipitate from the solution
51. 6:58For this will perform another step of centrifugation
52. 7:02This time we'll centrifuge at higher speed. Actually you can use the maximum speed of the centrifuge for this
53. 7:10So in our case, I will centrifuge for five minutes at fourteen thousand six hundred RPM
54. 7:23So now our pellets are ready, and we can continue
55. 7:30as you can see
56. 7:33centrifugation separated very well the solution from the pellet and in the pellet we'll have all the lipids
57. 7:39and the Membrane and the chromosomal DNA all precipitated on the bottom
58. 7:46and now we're going to proceed with the
59. 7:49for the next step for this we'll need a collection tube and
60. 7:56The filter column they are usually provided with a kit
61. 8:02We will have to pour the supernatant on top of the column
62. 8:08So the supernatant now contains our plasmid in the solution
63. 8:25You can now discard the pellet and proceed
64. 8:31And proceed with a with a column
65. 8:35We will now centrifuge the column one minute at maximum speed
66. 8:44To let the solution flow through the filter
67. 8:53Now that we have let the flow through get through the column we can discard
68. 9:00The flow-through and replace the same column into the same collection Tube. We will now wash the filter
69. 9:08with a wash solution containing ethanol
70. 9:15This will keep the DNA
71. 9:16bound to the column, but it will allow the rest of the
72. 9:21contaminants go into the waste tube down here
73. 9:27So we'll wash twice with 500 microliters of wash buffer
74. 9:49Once again, we centrifuge one minutes at maximum speed
75. 9:57So we now repeat the wash with the same amount of wash buffer
76. 10:03So once again we discard the flow through
77. 10:07We replace the column
78. 10:11And we have to add the wash buffer
79. 10:41So now once again. We discard the flow-through and
80. 10:45Now I'll have to
81. 10:47Dry the column to avoid all the ethanol present
82. 10:55For this, we'll centrifuge two minutes at maximum speed without any solution added to the column
83. 11:06Good. At this step it's essential to make sure that there is no ethanol left in the tube
84. 11:13So we just keep the column. And if you want to check if there is still ethanol left
85. 11:18You can flick the tube a little bit with your fingers
86. 11:24There's no droplets left that means that the column is ethanol free we can now take a clean
87. 11:311.5 mL Tube
88. 11:33in which we will collect our DNA
89. 11:36So place the filter on top of the tube
90. 11:41You can wait a minute or two just to make sure that all the residual ethanol evaporates or it can go straight ahead
91. 11:48So to collect our plasmid DNA we will now have to add
92. 11:53the elution buffer or you can use just a nuclease pure water
93. 11:58nuclease free water
94. 12:06The volume to add depends on the concentration you want to get into in the end. If you want a really concentrated sample
95. 12:13you're interested in putting as little water as you as you can. The minimum volume is about 35
96. 12:20microliters, but you can get up to 100 microliters if you don't care about the concentration so much.
97. 12:27I usually do something in the between
98. 12:30So I will put 40 microliters of nuclease free water
99. 12:43At this step it's important to pour the water
100. 12:47right in the middle of the of the column and not to touch the filter with the tip
101. 12:52So I pour the water and now we keep the column like this for a couple of minutes to wait
102. 13:02until the water soaks the filter and all the DNA comes from the filter into the solution
103. 13:16Now we can elute the DNA into our clean tube
104. 13:24For this once again, we will centrifuge one minute at maximum speed
105. 13:42so at this point our protocol is finished and
106. 13:45you can discard the column and
107. 13:48You have your DNA miniprep.
108. 13:51Now the only thing that's left is to label the tube correctly
109. 14:00So I will put the name of the plasmid and the date
110. 14:08the best way to label is to put the name of the plasmid both on the tube lid,
111. 14:15and on the side. And now we can check the concentration of the DNA by a spectrophotometer.