Endnote Cheet Sheet

EndNote Cheet Sheet

[fav]

Arabidopsis Seed Collection

[fav]

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

[fav]
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)

Inmproving Infection Efficiency Via Virus Centrifugation

1. Ultracentrifuge the virus at 50,000 x g for 90 min at 4°C.
Remove the supernatant. Drain carefully and well (preferably with a pipet) since the viral pellet is glassy and will be a filmy smear on the side of the tube.
 
2. Resuspend the virus to 0.5–1% of the original volume in TNE and incubate overnight at 4oC. Swirling during incubation may damage the virus. Gently pipet the solution to mix only after the overnight incubation, to allow diffusion of the virus. TNE is Tris buffer with NaCl and EDTA, which helps maintain the pH and is appropriate for storage if desired. Media can be used if the virus will be used immediately.
 
3. If desired, perform a second round of ultracentrifugation (Steps 1–2) by pooling previously concentrated virus.
 
4. This step is necessary only if injecting into animals, not if infecting cells in culture: Remove cellular debris and aggregated virus by low speed centrifugation (500 x g) for 5 min at 4oC.
 
5. Determine the viral titers of pre- and post-concentrated viral supernatants.
 
6. Infect target cells according to the Retroviral Gene Transfer and Expression

Immunofluorescence Double Staining Protocol Parallel Approach

1. Preparation of Slides

 

 A. Cell Lines

  • Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C

  •  Wash briefly with PBS
  • Fix as desired. Possible procedures include:

    10 minutes with 10% formalin in PBS (keep wet)

    5 minutes with ice cold methanol, allow to air dry

    5 minutes with ice cold acetone, allow to air dry

  • Wash in PBS

 B. FrozenSections

 

  • Snap frozen fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at – 80 ºC.

  • Cut 4-8 um thick cryostat sections and mount on superfrost plus slides or gelatin coated slides. Store slides at – 80 ºC until needed.
  •  Before staining, warm slides at room temperature for 30 minutes and fix in ice cold acetone for 5 minutes. Air dry for 30 minutes.
  •  Wash in PBS

 C. Paraffin Sections

  • Deparaffinize sections in xylene, 2x5min.

  • Hydrate with 100% ethanol, 2x3min.
  •  Hydrate with 95% ethanol, 1min.
  •  Rinse in distilled water.
  •  Follow procedure for pretreatment as required.

2. Pretreatments of Tissue Sections

 

Antigenic determinants masked by formalin-fixation and paraffin-embedding often may be exposed by epitope umasking, enzymatic digestion or saponin, etc. Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded.

 

3. Procedure

 

Note: prior to perform double labeling, it is important to test each primary antibody individually and select the best pretreatment(s) for each antibody. It will be ideal if the two primary antibodies require same pretreatment. Otherwise, one should do a further test by treating sections with both pretreatments and then immunostain for each antibody individually. If both antibodies survive the “double pretreatments”, you are ready for immunohistochemistry double staining. Another alternative is to do pretreatments separately for each antibody staining.

  1.  Rinse Sections in PBS-Tween 20 for 2×2 min.

  2. Serum Blocking: incubate sections in normal serum blocking solution – species same as secondary antibody (for example: primary antibodies are mouse and rabbit, and secondary antibodies are horse anti-mouse, and goat anti-rabbit, so horse and goat serum block should be used).
  3. Primary Antibodies: incubate sections in the mixture of two primary antibodies (mouse and rabbit) at appropriate dilution in antibody dilution buffer for 1 hour at room temperature.
  4. Rinse in PBS-Tween 20 for 3×2 min.
  5. Secondary Antibodies: incubate sections in the mixture of two fluorescent conjugated secondary antibodies (FITC conjugated Horse anti-Mouse and Texas Red conjugated Goat anti-Rabbit) in PBS for 30 minutes at room temperature).
  6. Rinse in PBS-Tween 20 for 3×2 min.
  7. Counterstain with DAPI if desired for 20 minutes at room temperature.
  8. Rinse in PBS-Tween 20 for 3×2 min.
  9. Coverslip with anti-fade fluorescent mounting medium and seal with nail polish.
  10. Store slides in dark at 4 °C.

 4. Results:

  •  1st Primary Antibody Staining Sites ——————- green
  •  2nd Primary Antibody Staining Sites——————- red  
  • Double Staining Sites ———————————– yellow
  • Counterstained Nuclei ———————————- light blue

Protocol For Cryosectioning

While the timing of the various steps in this protocol are probably not critical, I tend to prefer to process the tissue all at once to ensure that RNA and/or proteins do not get degraded.

 

Solutions

20% Paraformaldehyde/4% Paraformaldehyde-PBS

200 g paraformaldehyde

1 ml 10N NaOH

up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°.

Mix 100 ml 20% Paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q

filter, and store at 4° for up to 2 weeks

 

Sucrose/PBS

30% sucrose 150 g sucrose

up to 500 ml with 1X PBS

filter sterilize and store at room temperature

 

 

Procedure

• Dissect and fix the tissue in fresh 4% paraformaldehyde on ice for 5-10 minutes.

• Wash for 5 minutes in 1X PBS and repeat.

• Transfer to 30% sucrose until the tissue sinks (5-10 minutes depending on the size of the tissue).

• Transfer through a 1:1 mixture of OCT:sucrose and then into OCT.

• Place the tissue in the cryomold, overlay with OCT, orient and freeze quickly on dry ice.

• Once the tissue is in the mold with OCT it should be oriented and frozen quickly because a film can form on the top of the mold (where the OCT is exposed to air) and make moving the tissue difficult.

• If the size of the mold is small enough place each block into an eppendorf tube and store at -80°C.

• Cut 20 micron sections and place on silinized superfrost slides (Protocol S.6).

• For best results, proceed immediately with immunohistochemical staining.

Purification Of Mitochondrial Dna

The basic procedure for breaking cells with glass beads as described in the reference Lang et al. (1977) is as follows.


 

MtDNA from Protists

A minimum of 2-3 g wet weight of cells are necessary for one DNA extraction (preferably 10-20 g). The cells are harvested in the early stationary phase by centrifugation. After resuspension in a sorbitol buffer (0.6 M sorbitol, 5 mM EDTA, 50 mM Tris pH 7.4), the cells are broken mechanically by shaking with glass beads, and a crude mitochondrial fraction is isolated by differential centrifugation. The mitochondrial fraction is lysed in the presence of 1% SDS and 100 ug/ml proteinase K, at 50 degrees Celsius for 1 hr. SDS is eliminated from the lysate by addition of 1 M NaCl; after 1 hr on ice, the precipitate (SDS protein complex) is removed by centrifugation. The total nucleic acids are fractionated on a CsCl gradient (1.1 g/ml, 40 000 rpm for 48 hours) in the presence of 10 ug/ml bis-benzimide (Hoechst 33258, Serva). The upper band (A+T- rich DNA) in the gradient consists in many instances of mtDNA, and is extracted and re-centrifuged in one or two subsequent CsCl gradients. A final yield of 0.3 – 5 ug mtDNA can be expected.


 

MtDNA from Fungi

The protocol is essentially the same as described above. However, many fungi produce cells with large vacuoles and it is often necessary to start from 10-30 g wet weight cells in order to obtain a few ug of mtDNA. 
When a fungus produces mycelia it can be alternatively ground in liquid nitrogen and the total nucleic acids extracted with guanidine hydrochloride (Deeley et al., 1977), except that we use a 6M guanidine-HCl solution for the lysis.

Screening And Profiling Protein Expression In Human Cancer Serum Using Antibody Array Technologies

Antibody arrays have been a promising and inexpensive tool for bulk analysis
of protein level changes in human plasma and serum. These analyses have
lead to proteomic profiling of a number of disease states, as well as biomarker
discovery. Here, we show the value of using a series of Panorama® antibody
microarrays comparing normal and cancer serum samples to identify potential
disease biomarkers. The arrays chosen for this work contained antibodies for
proteins with known significance in intracellular processes. These arrays were
used because it is believed that biomarkers exist in the low abundance tissue
leakage proteins that make up approximately one-third to one-half of the
thousands of proteins found in blood. In our study, we have also highlighted
the contribution that depletion gives to the discovery/validation of the low
abundance tissue leakage proteins on the antibody microarrays.
 
Materials and Methods
Serum Samples
Serum samples were obtained through Genomics Collaborative. Cancer samples
were from either hepatocellular carcinoma (36-year old Vietnamese male) or
renal cell carcinoma (66-year old Caucasian male) patients. Normal samples
were from a 51-year old Caucasian male.
Serum Depletion
Depletion was completed using the ProteoPrep® 20 Immunoaffinity Depletion
Kit (PROT20) and following the supplied protocol.
ELISA Analysis
Serum samples were coated onto 96-well ELISA plates. Plates were incubated
with purified primary antibodies corresponding to those on the arrays. Plates
were washed and incubated with HRP-conjugated secondary antibodies. After
a final wash, the plates were visualized using TMB substrate, and stopped with
1 M HCl. The absorbance was measured at 450 nm.
Serum samples were first depleted of twenty of the high abundance proteins.
The depleted samples were then labeled with either Cy3 or Cy5 (Amersham)
and mixed at equal protein amounts to allow for parallel analysis. The labeled
serum samples were incubated on both the Panorama Antibody Array – p53
Pathways (PPAA4) and the Panorama Antibody Array – Cell Signaling (CSAA1)
for 30 minutes. Following incubation, the slides were scanned using a ScanArray
Express (Perkin Elmer) and analyzed using ImaGene 7.0 software (BioDiscovery).
Each Panorama p53 array was incubated with 100 μg of both depleted and
whole (non-depleted) serum conjugated with either Cy3 or Cy5. A dye swap
was performed to confirm results. In the comparisons, the whole serum is green
and the depleted serum is red. Select proteins are identified. Note that only the
top half of the Cell Signaling array slide is shown. As seen in the comparisons
above, more proteins are visible in the depleted serum than in the whole serum.
The depletion has made the less abundant proteins visible.
 
Following depletion, each Panorama p53 array was simultaneously incubated
with a mixture containing equal amounts of normal and cancer serums (Renal
cancer sample for slide A, Liver cancer for slide B) conjugated with either Cy3
or Cy5. A dye swap was performed to validate results. As seen in the slides, a
number of spots were differentially expressed with the cancer samples when
compared to the normal control. In both comparisons above, the normal serums
are labeled red and the cancer serums are labeled green. Therefore, a red spot
would indicate down-regulation in the cancer sample, and a green spot would
indicate up-regulation in the cancer sample. Select proteins are identified.
Similar results were seen using the Cell Signaling array. Additional proteins
found to be significantly different using the Cell Signaling array include, but
are not limited to: alpha Catenin (up-regulated in both cancer samples), MAP
Kinase (Erk1 + Erk2) (up-regulated in both cancer samples), Calmodulin (upregulated
in the liver cancer sample), Cyclin D1 (down-regulated in the liver
cancer sample), DOPA Decarboxylase (down-regulated in both cancer samples),
and Synculein (down-regulated in the renal cancer sample). 

Expression Levels Of The Predicted Proteins By Western Blotting

To evaluate the prediction accuracy of DAMAPEP, expression levels of the 10 proteins predicted with the 2.0 cutoff value were examined by Western blotting. Among the seven proteins predicted with increased expression in cancer cells, five proteins, RAIDD, Rb p107, Rb p130, SRF, and Tyk2, were confirmed to have higher expression (Fig. 4a). For the other five proteins predicted with DAMAPEP, increased expression level of leukemia inhibitory receptor factor alpha was only detected in some cancer cell lines. For c-Kit and IL-2Rβ, no bands were observed in Western blots in any cell lines probably due to extremely low expression levels. The expression levels of Mos proto-oncogene and IκB-β were similar in both normal and cancer cell lines (data not shown). The expression of RAIDD was almost undetectable in the three normal cell lines. The expression of Rb p107, SRF, and Tyk2 in normal cells was between 15 and 40% of their expression in cancer cells. The expression of Rb p130 in normal cells was about 20% of its expression in cancer cells (Fig. 4b). Mean values of the normalized expression of RAIDD, Rb p107, Rb p130, SRF, and Tyk2 in three normal cell lines are 5 versus 61% (p = 0.0032), 26 versus 81% (p = 0.0041), 18 versus 69% (p = 0.0055), 25 versus 73% (p = 0.0033), and 30% (p = 0.0012), respectively (Fig. 4c). Those data confirm that RAIDD, Rb p107, Rb p130, SRF, and Tyk2 are indeed overexpressed in the tested cancer cells.

FIG. 4.

Validation of DAMAPEP-predicted resultsa, Western blotting confirms that five proteins have higher expression levels in cancer cells. Total cell extracts of the 10 breast cell lines were prepared as described under “Experimental Procedures.” Equal amounts of total lysates, as shown for actin, were loaded for Western blotting analysis. Proteins are labeled at the left of the gel, and tested cell lines are labeled at the topN1N2, and N3 represent three normal cell lines, ER+1ER+2, and ER+3 represent three estrogen receptor-positive carcinoma cell lines, and ER−1ER−2ER−3, and ER−4 represent four estrogen receptor-negative carcinoma cell lines. b, relative expression of five proteins in 10 breast cell lines. Western blot analysis for every protein was repeated at least three times. The average values for every protein in different cell lines are shown here with error bars corresponding to their S.D. c, box plot of the relative expression of five proteins in three normal cell lines and seven cancer cell lines using the data shown in b. The ranges of expression levels in the group of normal cell lines versus the group of cancer cell lines are shown side by side for every protein. The interquartile ranges are shown by boxes with the median values in filled trianglesN, normal;C, cancer; HQ, higher quartile; LQ, lower quartile.

 

 

Identifying The Differentially Expressed Proteins Between Normal And Cancer Cell Lines By Damapep

The expression profiles of 312 proteins obtained by the DAMA staining for 10 breast cell lines were repeated at least twice and were analyzed by the programs ScanAlyze and DAMAPEP. When using a minimum cutoff value of 2.0 for the absolute value of Ratio(ij), 10 of 312 proteins (leukemia inhibitory factor receptor alpha, Tyk2, Rb p130, SRF, c-Kit, Rb p107, RAIDD, Mos proto-oncogene, IκB-β, and IL-2Rβ) were predicted by DAMAPEP to have different expression levels in cancer cells versus normal cells (Fig. 3a). The first seven proteins were predicted to have higher expression levels in cancer cells, and the latter three were predicted to have lower expression levels. Their corresponding dots in the DAMA images for all 10 cell lines demonstrated the consistency between the DAMAPEP prediction and the original DAMA staining data especially for those confirmed proteins (Fig. 3b and supplemental Fig. S1). When the cutoff value for Ratio(ij) was decreased from 2.0 to 1.5 (representing a 50% intensity change between normal and cancer cells) six extra proteins were added to the prediction list. Among them, ErbB3, c-Raf, and Rad52 were predicted to have higher expression in cancer cells, and Fas (TNFRSF6)-associated death domain protein, FLICE inhibitory protein (short/long), and IL-1R were predicted to have lower expression. However, none of these six proteins was confirmed to have the predicted difference by Western blotting analysis (data not shown). Therefore, 2.0 was used as the cutoff value for Ratio(ij) in the DAMAPEP prediction.

FIG. 3.

Proteins predicted from DAMAPEP to have different expression levels between normal and cancer cellsa, the DAMAPEP-predicted proteins are shown with their name, position in the DAMA staining images, and their corresponding ratio values ranked from high at left to low at rightb, summary of the corresponding dots in the DAMA staining images shown in Fig. 1 for the seven proteins predicted by DAMAPEP with increased expression levels in carcinoma-originated cell lines. LIFR, leukemia inhibitory factor receptor alpha.