2022 (Arabidopsis)

2022 (Arabidopsis) kdorfman Wed, 12/22/2021 - 17:40

Planting schedule

Week Day Date Lab
1 W 1/26 0.1: Plant Col0 seeds (24 pots),
micropipetting
2 M 1/31 0.2: Benchling platform
1.1: Initial working maps
W 2/02 1.2: Finalizing gene models
3 M 2/07 1.3: Identifying T-DNA insertion sites
W 2/09 1.4: Designing primers
4 M 2/14 2.1: DNA extraction (plants from week 1)
Lab 1 report due
W 2/16 2.2: DNA quantification
5 Tu 2/22 4.1: BLAST
W 2/ 23 4.2: Literature search
6 M 2/28 2.3: Testing PCR conditions
W 3/02 0.3: Arabidopsis essentials (6 rose pots per table)
3.1: Salk like genetics
plant Salk seeds (48 pots)
Lab 2 report due
7 M 3/07 4.3: Phylogeny
W 3/09 Catch-up Day
SPRING BREAK
8 M 3/21 3.2: Phenotyping Salk mutants
W 3/23 5.1: Genotype Salk mutants (DNA extraction)
Lab 4 report due
9 M 3/28 5.2 Genotype Salk mutants (PCR)
W 3/30 5.3 Genotype Salk mutants (gel purification)
10 M 4/04 5.4: Sequence analysis
W 4/06 6.1: Gene expression resources
Lab 5 report due
11 M 4/11 6.2: Statistical analysis of gene expression data
W 4/13 6.3: q-RT-PCR planning (see Planting schedule for prep)
12 W 4/20 6.4: RNA extraction and quantification
13 M 4/25 6.5: RT and qPCR
W 4/27 catch-up day
Lab 3 report due
14 M 5/02 6.6: Analysis of qPCR data
W 5/04 Work on final presentation
Finals M 5/09 Lab 6 report due
W 5/11 Final presentation due
Last chance for make-ups
Extra credit due

2.1 DNA extraction 2022

2.1 DNA extraction 2022 kdorfman Thu, 02/10/2022 - 14:21

Here are the equipment & reagents

LN2, tongs and dewars

ball mill (pre-chill the blocks)

grinding tubes and steel balls

miracloth cut into squares

funnels

Compost pot

Stinky waste bucket in hood for Beta mercapto ethanol

Dirty pots dishpan

Reagent per rxn per pair per class
DNA extraction buffer 600 uL 1250 uL 16.25 mL
KOAc 5M </a. 250 uL 550 uL 7.150 mL
Isopropanol 600 uL 1200 uL 15.6 mL - pre-aliquoted into round bottom centrifuge tubes
EtOH 70% 400 uL 850 uL 12 mL
NaOAc 3M 10 uL 25 uL 325 uL
T10E5 100 uL 225uL 3 mL
EtOH 100% 200 uL 450 uL 6 mL
T10E1 50 uL 125 uL 1.625 mL
2022 DNA extraction lab manual

5.1 DNA extraction for genotyping

5.1 DNA extraction for genotyping kdorfman Tue, 03/22/2022 - 13:11

From Elizabeth Vierling

Per Reaction:

  • 500 uL Edwards Extraction Buffer (6.5 mL/pair, 100 mL/class)

  • 300 uL Isopropanol (3.9 mL/pair, 50 mL/class)

  • 100 uL T10E1 (1.3 mL/pair, 20 mL/class)

  • 1 2-mL grinding tube (12 tubes/pair, 50/class)

  • 2 steel beads (24 beads/pair, 300 beads/class)

Per class * Dresch ball mill grinder

  • 6 microcentrifuge

  • 6 vortex

Edwards DNA Extraction 2022-1-7.docx

5.2 Genotyping Salk mutants (PCR)

5.2 Genotyping Salk mutants (PCR) kdorfman Thu, 03/24/2022 - 18:15

Check extracts for DNA

  • 1% gels
  • HW Massruler

Amplify extracted DNA 24 PCR reactions per pair (12 WT + 12 Salk)

Reagent WT allele master mix uL mutant master mix uL per pair per class
H2O 243.25 243.25 486.5 ~6 mL
10X ex taq buf 35 35 70 ~145 uL
dNTP 28 28 60 720 uL
LBP 17.5 17.5 210 uL
GSP 1 17.5 ?
GSP 2 17.5 ?
Taq 5U/uL 1.75 uL 1.75 uL 3.5 uL 42 uL

6.4 RNA extraction 2022

6.4 RNA extraction 2022 kdorfman Tue, 04/19/2022 - 14:18

RNA Extraction

Materials for the class:

  • 6 centrifuges
  • Tissuelyzer blocks in the -80
  • LN2
  • Freezer box to keep RNA in the -80

Materials per group:

  • scissors
  • ice bucket
  • jeweler's scale
  • 6 grinding tubes with 2 metal beads each
  • 6 lilac columns
  • 6 pink columns
  • 6 capless collection tubes

Reagents per group:

  • ~3 mL RLT + β-Mercaptoethanol
  • 1.5 mL 95% Ethanol
  • 4.2 mL buffer RW1
  • 6 10 uL aliquots DNAse
  • 420 uL buffer RDD
  • 6 mL buffer RPE
  • 300 uL RNase free water

6.5 RT qPCR

6.5 RT qPCR kdorfman Thu, 04/21/2022 - 20:08

One-step RT-qPCR

One-step RT-qPCR kdorfman Tue, 04/26/2022 - 14:18

Luna Universal One-Step RT-qPCR

NEB E3005S, 200 rxns

  • Prepare RNA of interest using desired RNA extraction and purification methods. Determine concentration by OD260 absorbance.
  • Make dilutions of RNA to be used for the standard curve. These should be prepared fresh before each experiment and can be diluted in either water or TE.

Reaction Setup: For best results, we recommend running each RNA standard and sample in triplicate.

COMPONENT 20 µl REACTION FINAL CONC
Luna Universal One-Step Reaction Mix (2X) 10 µl 1X
Luna WarmStart® RT Enzyme Mix (20X) 1 µl 1X
Forward primer (10 µM) 0.8 µl 0.4 µM
Reverse primer (10 µM) 0.8 µl 0.4 µM
Template RNA variable < 1 µg (total RNA)
Nuclease-free Water to 20 µl

  1. Thaw Luna Universal One-Step Reaction Mix and other reaction components at room temperature, then place on ice. After thawing completely, briefly mix each component by inversion, pipetting or gentle vortexing.

  2. Determine the total volume for the appropriate number of reactions, plus 10% overage and prepare assay mix of all components except RNA template accordingly. Mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.

  3. Aliquot assay mix into qPCR tubes or plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.

  4. Add RNA template to qPCR tubes or plate. Seal tubes with flat, optically transparent caps; seal plates with optically transparent film. Care should be taken to properly seal plate edges and corners to prevent artifacts caused by evaporation.

  5. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).

  6. Program real-time instrument with indicated thermocycling protocol (see table below). Ensure a plate read is included at the end of the extension step.

CYCLE STEP TEMP TIME CYCLES
Reverse Transcription 55°C1 10 minutes 1
Initial Denaturation 95°C 1 minute 1
Denaturation
Extension		 95°C
60°C		 10 sec
30 sec2 (+ read)		 40-45
Melt Curve 60-95°C3 various 1

Notes

  1. A 55°C RT step temperature is optimal for Luna WarmStart Reverse Transcriptase. To ensure best performance and full WarmStart activation, avoid using a temperature of < 50°C. ↩︎

  2. For Applied Biosystems real-time instruments use a 60 second extension step. ↩︎

  3. Follow real-time instrument recommendations for melt curve step. ↩︎

RT, qPCR

RT, qPCR kdorfman Tue, 04/26/2022 - 14:16

Separate RT & qPCR

RT 10 groups @ 6 rxns 2 groups @ 8 rxns

Equipment

Hot blocks at

  • 50°C (2 dry baths - this is the long step and there are at least 4x as many tubes as groups),
  • 65°C
  • 70°C
    reagent uL/rxn uL/8 rxn (+ xtra) uL/class notes
    Oligo dT 50 µM 1 1 15 180
    10 mM dNTP mix 2 1 15 180
    RNAse-free water 11 + 40 60 720 aliquot once for both RT and qPCR
    5X 1st strand buffer 4 60 720 comes with Superscript
    0.1M DTT 1 15 125 1500 | comes with Superscript
    RNaseOUT 3 1 8 96 Dispensed from freezer box during lab
    Superscript 4 1 8 96 Dispensed from freezer box during lab

864 rxns @ 10uL

(24 x 3 rxns per group x 12 groups)

Reagent uL per rxn uL per 75 rxns
2x SYBR Green master mix5 5 uL 375 uL
water <5 uL 375 uL
forward primer 0.5 uL
reverse primer 0.5 uL
cDNA ~1 uL

  1. Fisher FERSO131 Oligo dT 100 µM (=0.5µg/µL) 60 µL @ ~$50. Dilute to 50µM!! (for emergencies: 50 µM oligo(dT) Fisher stockroom: C1101 0.5µg/µL x 40µL = 20µg) - Order T(18) from Invitrogen??? ↩︎

  2. 14 µL each + 84 µL RNase-free water OR Fisher FERR0192 ↩︎

  3. Invitrogen 10777019 RNAse out 5KU @ $143 ↩︎

  4. superscript III 200U/µL Invitrogen 18080044 10KU @ $259

    qPCR ↩︎

  5. Thermo Fisher 4385612 ↩︎

Gel purification

Gel purification kdorfman Tue, 03/29/2022 - 16:21

Zymoclean Gel DNA Recovery Kit Zymo Research D4002

  • Add 26 mL 95% EtOH to the 6 mL DNA Wash Buffer concentrate
  • Excise DNA fragment (x-tracta Gel Extraction Tool or razor blade)
  • Add 3X volume ADB (OR 300 uL ADB/mg gel)
  • Incubate 37-55C 5-10 minutes or until gel is dissolved
  • transfer melted agarose solution to Zymo Spin Column in a Collection Tube
  • centrifuge 10 - 16K Xg 30-60 sec
  • add 200 uL DNA Wash Buffer
  • Discard flow-through
  • Repeat with another 200 uL DNA Wash Buffer. Discard flow-through again
  • Add >= 6 uL DNA Elution Buffer (or water)
  • Put into clean 1.5 mL tube
  • Spin 30-60 sec to elute DNA.

Per rxn:

Reagent amount aliquot for 2 rxns for 3 rxns
ADB1 300 uL 640 uL 950 uL
DNA Wash Buffer 400 uL 840 uL 1250 uL
ENA Elution Buffer 6 uL 15 uL 22 uL (or water?)
  • Jewelry scale to weigh gel
  • Hot block 55 C for incubation and melting of agarose
  • gel excision tools
  • 2 mL tube for melting gel
  • vortex

  1. volume depends on size of excised gel piece ↩︎

Zymoclean Gel DNA Recovery Kit protocol

Planting schedule 2022

Planting schedule 2022 kdorfman Thu, 12/23/2021 - 15:54

Pots for students to plant seeds for genomic DNA

12 pots for Week 1 (1/26/22)

Planting for Week 5:

Arabidpsis essentials (2/23/22):

  • 2 seeds in each of 6 rose pots every week,
  • starting 1/12 (2 week before classes begin),
  • for a total of 36 pots
  • 6 1/2 MS plates 1 week before week 5 (seeds to fridge on 2/14/22)

Students plant mutant seeds:

  • 12 groups, 2 pots mutants, 2 pots Col0
  • for a total of 48 3" pots

Planting for Week 11:Gene expression planning:

Seeds on plates for RNA low and high iron experiments: ? at least 12 1/2 MS plates, 6 plates low iron.

5 seeds in each of 24 pots every week for 4 weeks, starting 6 weeks before week 11. (weeks 7, spring break, 8, and 9), for a total of 96 pots

1 row of seeds on each of 24 plates, 1 week before week 11

Date plant for
1/12 6 rose pots 6 week old Arabidopsis
1/19 6 rose pots 5 week old Arabidopsis
1/26 6 rose pots 4 week old Arabidopsis
2/02 6 rose pots 3 week old Arabidopsis
2/09 6 rose pots 2 week old Arabidopsis
2/16 6 1/2 MS plates 1 week old Arabidopsis
2/23
3/01 12 1/2 MS plates seedlings on cellophane for cDNA (iron expt)
3/03 plates to growth chamber start seedlings growing
3/07 24 pots 6 week old for gene expression
3/11 txfer plated seedlings half to MS, half to low iron
3/14 harvest iron expt seedlings Stavroula makes cDNA
3/14 24 pots 5 week old for gene expression
3/21 24 pots 4 week old for gene expression
3/28 24 pots 3 week old for gene expression
4/06 24 plates 2 week old for gene expression
4/13 24 plates 1 week old for gene expression