Endnote Cheet Sheet

EndNote Cheet Sheet

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Arabidopsis Seed Collection

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Arabidopsis Seed Collection

Luca Comai has devised a simple seed collector suitable for high density use (many plants in a small space). It is effective, very cheap, and easy to construct. The instructions for constructing it are given below. They may sound complicated but the collector is really very simple. Feel free to ask questions or send comments.

 

Seed collector II

Introduction

This seed collector is suited for harvesting seed from individual plants grown in small pots (e.g.: square, 6 cm wide at top, tapered to 4.5 cm at bottom, 8 cm high). Each pot can be placed next to other pots to achieve high density spacing.

 

Materials

  1. Adhesive tape, such as VWR or Time-Med pressure sensitive labeling tape 1.8 cm wide. The tape type is not important as long as it sticks and it holds up to greenhouse conditions
  2. Paper stapler
  3. Plastic film, such as Mylar overhead transparency film (0.002 mil, Vu-Color ). The choice of this film is based on characteristics and availability. We recycle the film used for lecturing. Other types maybe suitable: experimenting is the best way to find out whether the film has the right flexibility. Mylar has one disadvantage: it builds an electrostatic charge that attracts seeds. However, in our case, the film comes for free and it looks pretty with all the lecture notes. We prefer instructors who use multicolored pens.

Construction

  1. Cut sheets 12 cm x 42 cm. Roll them lengthwise on a dowel 33 mm in diameter and 50 cm long. The long sides will overlap by about 1.3 cm
  2. Tape the resulting plastic tube once, at one third the distance from one extremity. Make a continuous ring of tape for maximum strength
  3. Flatten and fold back the end of the tube most distant from the tape, in such a way that the seam is central and internal to the fold. Staple the sides just above the fold. The fold line should be 2 cm from the end
  4. About 9 cm above the fold, make a cut perpendicular to the tube. The cut will comprise half or slightly less than half of the circumference and will place the seam in the center of the cut. The collector is finished
  5. Appress the flattened end of the collector to the side of the pot. Place the cut about 5 cm above the rim of the pot and facing the pot. Tape the collector to the pot with a full ring of tape
  6. Gently spread the cut and introduce the young inflorescence. As secondary inflorescences are produced guide them in the collector or remove them
  7. A plant with a fully developed inflorescence can be easily fitted with a collector.
    • Gently lay the pot on its side so that the inflorescence fits over half a sheet of standard printer paper (7.5 cm x 26 cm)
    • Roll the paper to enclose the inflorescence. Make the roll’s diameter smaller than the collector’s. Tape the roll to avoid unfolding and pull it to where the length of the inflorescence-paper roll assembly is longer than that of the collector
    • Place the pot at the edge of a table with the inflorescence leaning out. Gently push the rolled inflorescence into the collector. Once fully inserted, the tip of the paper roll should stick out or be easily reached. Pull the paper roll out, leaving the inflorescence in the collector
    • Tape the collector as in “5”

Comments

The dimension of the collector can be changed to fit any square pot. Its design can also be modified to fit special situations. We harvest the seed by cutting the inflorescence at its base, throwing the label into the collector and closing its ends. Collectors can be washed and reassembled or thrown away. I would appreciate knowing of any improvement. For big pots and when space is not a limitation, our previous seed collector (see Compleat guide, AATDB) works well.

Dna Extraction Procedure From Human Blood

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1. Draw 5 ml of blood using lavender-top Vacutainer (Beckton-Dickinson; EDTA anticoagulant)
2. Keep cool until prep is performed (e.g. in an ice chest), but DO NOT FREEZE. Highest yield will be achieved by extracting within 24 hours.
3. Empty blood into a 15 ml Falcon tube; add 10 ml of Red Cell Lysis Buffer (RCLB) and mix completely by inversion.
RCLB: 1 mM NH4HCO3
115 mM NH4Cl
Note: You can bring pre-weighed NH4Cl and a 1M solution of NH4HCO3, and use the cleanest water available to make RCLB on-site. Try to use distilled water, but in a pinch I suspect that water from a personal filter (e.g. Pur, etc.) would work. If it is to be used immediately, this solution does not need to be autoclaved; if you plan to store it for more than a day, autoclave.
4. Spin 10 minutes @ 1,200g in a clinical centrifuge.
5. Discard supernatant and resuspend cell pellet in 10 ml RCLB; repeat step 4.
6. Discard supernatant; resuspend cell pellet (it should be white now) in 1.8 ml of White Cell Lysis Buffer (WCLB). It should be extremely gelatinous – like snot.
WCLB: 100 mM Tris-Cl (pH 7.6)
40 mM EDTA (pH 8.0)
50 mM NaCl
0.2% SDS
0.05% Sodium azide
Note: This solution (before the addition of SDS) definitely needs to be autoclaved – this means bringing a heavy, well-protected bottle with you, or being completely certain that you will be able to autoclave on site. Add SDS after the autoclaved solution cools. Remember that the DNA is going to sit around in this stuff under less-than-ideal conditions for a considerable period of time…
7. Store each white cell lysate in a 2 ml screw-top tube. DNA should be stable in this form for several weeks at room temperature, although refrigeration is recommended just to be safe.
8. To perform final extraction, add 150 µl of saturated NaCl (~ 6M NaCl) to 400 µl of white cell lysate.
9. Vortex and invert to mix; place on ice for 10′.
12. Centrifuge 5′ @ 12,000 RPM.
12. Add the supernatant to a 1.5 ml tube (550 µl); add 1000 µl absolute (100%) ethanol. Mix by inversion – the DNA precipitate should be visible at this point.
13. Spin 5′ @ 12,000 RPM; discard supernatant.
14. Wash w/ 1 ml 70% ethanol; air dry and resuspend in 100 µl of dilute TE (1:4 w/ H2O). Leave overnight at 4°C to resuspend completely. Check concentration and dilute to 100 ng/µl for stock. Freeze white cell lysates at -70°C for long-term storage.
Recommended field equipment for DNA extraction
1. Clinical centrifuge capable of at least 1,000 RPM, or hand-cranked model
2. 10 ml pipettes (minimum of 1 per extraction session – e.g. 1/day)
3. 15 ml Falcon tubes (1 per sample)
4. P-1000 Pipetteman
5. 1000 µl pipette tips (minimum of 1/sample)
6. 2 ml screw-top tubes
7. Gloves
8. Tube racks (for Eppendorfs and 15 ml tubes)
9. Blood collecting supplies (Vacutainers, needles [21 gauge if possible], plastic housings, tourniquets, alcohol preps, cotton, Band-Aids, biohazard bin for needles)
10. Ice chest (pack the other supplies inside for travel – and bring documentation for customs in case you are asked about all of those needles, white powders and solutions inside)