LABORATORY Day 3:Isolation and Analysis of Recombinant Plasmid DNAs Containing Cloned 16S Sequences

Objectives of Laboratory 1D:

1. Purify plasmid DNA from liquid cultures of one white transformant

2. Cleave the purified DNA with the restriction endonuclease EcoRI

3. Analyze these DNAs using agarose gel electrophoresis

4. Observe your plates of cultured soil microbes

Flow Chart of Laboratory 1D:

Purify One Cleave DNA Analyze DNA usingObserve Cultured

Plasmid DNA With EcoRI Electrophoresis Soil Microbes

I. INTRODUCTION:Earlier this week, you isolated metagenomic DNA from your soil sample, used PCR to amplify 16S sequences from this sample and cloned them into pCR2.1. Today, you and your partner will each isolate and analyze plasmid DNAs from one of your white transformant before the purified DNAs are sent for nucleotide sequence determination.

A diagram of the vector pCR2.1, into which you cloned your amplified 16S genes, is shown above. Note that the polylinker has two recognition sequences/sites (arrows above) for the restriction enzyme EcoRI and that these two sites flank the region into which your fragment was inserted. Today, you will make use of the fortuitous locations of these EcoRI sites to analyze the structure of your recombinant plasmids.

II. EXPERIMENTAL PROCEDURES: Today you will use the QIAprep Miniprep kit to isolate plasmid DNA from one of the cultures you inoculated yesterday. This kit is widely used in our Department because it produces very pure yields of plasmid DNA that are suitable for sequencing and is also fast and relatively simple.
A. Isolation of Recombinant Plasmid DNA: Each person will isolate plasmid DNA following the procedure described below.
1. Obtain the two tubes containing the cultures you inoculated yesterday from the incubator. /  / Choose one of these tubes to use in isolating plasmid DNA.
2. Obtain a clear 1.7 ml microtube from the canisters on the front bench and label the top with your initials and the number of the culture you’ve chosen.
3. Use your P1000 to transfer 1.0 ml from your culture tube to the microtube.
4. Pellet the bacterial cells in this culture by spinning it at maximum speed for 1 min. /  / Be sure to place your tube directly across the rotor from another tube for balance.
5. Pour off the clear supernatant above the pellet into the sink.
6. Vortex vigorously for about 30 sec to resuspend the pelleted cells in the residual liquid.
7. Add 250 l Buffer #1 (tube #1, purple dot) to your tube and mix by vortexing vigorously. /  / Make sure no clumps of cells are present.
8. Add 250 l Buffer #2 (tube #2, red dot) to your tube and mix thoroughly by inverting the tube 4 – 6 times. Do not vortex. /  / The solution will turn blue.
9. Add 350 l Buffer #3 (tube #3, orange dot) to your tube and mix it immediately and thoroughly by inverting it several times. The addition of Buffer #3 should make the solution colorless. However, if any blue color or clumps remain, invert your tube to mix until they are no longer visible. /  / This buffer causes genomic DNA, which has a large molecular weight, to precipitate. The plasmid DNA, which is much smaller, stays in solution.
10. Centrifuge for 10 min at 13,000 rpm (maximum speed) in the microfuge. /  / A large white pellet of genomic DNA and debrisshould be visible.
11. Pour the supernatant (the solution above the pellet) in your tube into a QIAprep spin column. /  / Do not discard the supernatant. It contains your plasmid DNA.
12. Centrifuge your column for 30 – 60 sec at 13,000 rpm and discard the flow-through into the sink. /  / Use 13,000 rpm for all remaining centifugation steps.
13. Wash your QIAprep spin column by adding 0.75 ml buffer #4 (tube #4, blue dot) and centrifuging for 30 – 60 sec.
14. Discard the flow-though from your tube and centrifuge for an additional 1 min to remove residual wash buffer. /  / Discarding the flow-through is essential before spinning again.
15. Place your QIAprep column in a clean 1.5 ml microfuge tube and add 50 l sterile H2O (clear tube, blue dot) to the center of the QIAprep column.
16. Let your column stand for 1 min and then spin for 1 min.
17. After the spin ends, discard the column and close the top of your tube.
18. Spin your tube in the microfuge for a few seconds to bring the DNA solution to the bottom of the tubes.
B. Cleavage of your Purified Plasmid DNAs with EcoRI: In order to confirm that the plasmid DNA you have purified contains an inserted fragment before it is sent out for sequence analysis, you will cut an aliquot of your plasmid DNA with the restriction endonuclease EcoRI. /  / The name EcoRI is derived from the first letter of the Genus and first two letters of the species name of the source organism, which is E. coli, strain RY12.
1. Obtain an Isotherm and some ice before beginning.
2. Take your Isotherm and ice to the front bench to obtain a microtube containing EcoRI (pink tube in Stratacooler) and another containing 10X EcoRI buffer (pink tube, black dot). /  / Each restriction endonuclease has a specific buffer, which provides the correct reaction conditions required for its optimal activity.
3. Take these tubes to the microfuge and pulse spin them for a few sec to bring the contents to the bottom.
4. Use the plasmid DNA you just isolated to make the following digest in a small clear microtube by adding the components in the order listed below: /  / Label the top of each tube with the abbreviation of each transformant.
13 l sterile water (clear tube, blue dot)
2 l 10X EcoRI buffer (pink tube, black dot) /  / When adding small amounts of components, add them to the liquid
3 l plasmid DNA (flick the tube to mix these components) / already present in the tube and not onto the side of the tube.
2 l EcoRI enzyme (pink tube)
20 l total volume
5. Flick your tube with your fingers to mix it well and pulse-spin it in the microfuge.
6. Place your tubes in a foam “floatie” and incubate at 37o C for 30 - 40 min. /  / Return the rest of your plasmid
DNA to the front lab bench so it can be sent out for sequencing.
7. During this incubation, pre-run an e-gel as described next.
C.Pre-running Your E-Gel: Eight or nine people will share one 1.2% e-gel to analyze the digests of their plasmid DNAs. Follow the same procedure you have used previously (pp. 40 – 42 of this manual) to pre-run your e-gel.
D. Loading and Running Your E-Gel: Use both hands to remove the gel comb by gently lifting it and rolling it slowly toward you.
1. Obtain a sample of the 123 bp Ladder (yellow tube) and a sample of Lambda DNA cut with HindIII (clear tube, green dot) from the front bench. /  / Flick these tubes gently but thoroughly to mix well.
2. When the incubation is over, retrieve your tube and add 2 l loading dye (clear tube, purple dot) to it.
3. Flick your tube with your fingers to mix and pulse-spin it in the microfuge.
4. Load 20 µl of the 123 bp Ladder (yellow tube) into one end lane.
5. Load 20µl of the Lambda DNA cut with HindIII (clear tube, green dot) into the other end lane.
6. Each person should load 20 lof her/his digest in one of the other wells. /  / Be sure to record the number of the gel and the lane you are using.
7. The gel will run for 20 -30 min until the dye has migrated slightly more than halfway and then you will take a picture for your records. / 
E. Observing Your Cultured Soil Microbes: While your gel is running, count any new colonies of soil microbes that have grown since your last observation. Also take a digital picture of some or all of your plates for your records. /  / Record these numbers in your notebook.

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