Course Prep

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2013 genes

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2015 rosters, etc.

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Enzymes & kits

  • Takara Ex-Taq
    giant economy size: RR001B 4x 250U $590 at Clontech
    $843 at Fisher


Fisher equivalent has this buffer: Tris-HCl 100mM, pH 9, KCl 500 mM, MgCl2 15 mM. Takara buffer is 20 mM MgCl2

  • try this? cheap Taq at http://www.bulldog-bio.com/bioreadyrtaq.html

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

  • Invitrogen 10777019 RNAse out 5KU @ $143

  • Invitrogen RNAse A Check supply.

  • Qiagen 79254 DNAse
    includes RNase-free water and RDD for diluting the DNAse

  • Qiagin RNEasy 74904

    • if needed RW1 1053394
    • if needed RLT 79216
  • Quantifast 204054 400 rxns for q pcr

  • Zymo Clean & Concentrator kit

  • Oligo-dT Fisher FERSO131 Oligo dT 100 µM 60 µL @ ~$50. Dilute to 50µM!! (for emergencies: 50 µM oligo(dT) Fisher stockroom: C1101 0.5µg/µL x 40µL = 20µg)

link title

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2015 (S) Restriction list

Enzyme Cut Smart NEB 1 NEB 2 NEB 3 NEB 4
Aat2 100 10 50 50
Acc651 25 10 75 100
Acl1 100 10 10 10
AflII 100 50 100 10
Age1[^1] 75 100 75 25
AlwN1 100 10 100 50
Apal1 100 100 100 10
Asc1 100 10 10 10
Ava1 100 10 100 25
Ava2 100 50 75 10
BamH1-HF 100 100 50 10
BciVI 100 100 25 10
Bgl2 10 10 10 100
BmgB1 10 10 10 100
BsaI 100 75 75 100
BspD1 100 25 75 50
BspE1 10 10 10 100
BsrB1 100 50 100 100
BsrG1 25 25 100 100
Cla1 100 10 50 50
Dra1 100 75 75 50
Eag1 10 10 25 100
Ear1 100 50 10 10
EcoR1 50 25 100 50
EcoRV-HF 100 25 100 100
HaeIII 100 50 100 25
Hha1 100 25 100 100
Hind3 50 25 100 50
Hind3 50 25 100 50
HinP1I 100 100 100 100
Hpa1 100 10 75 25
Hpa2 100 100 50 10
Kas1 100 50 100 50
Mbo1 100 75 100 100
Mlu1 25 10 50 100
Msp1 100 75 100 50
Nae1 100 25 25 10
Nco1 100 100 100 100
Nde1 100 75 100 100
Nhe1-HF 100 100 25 10
Pst1 50 75 75 100
Pvu1 10 10 25 100
Pvu2 100 50 100 100
Rsa1 100 25 50 10
Sac1 100 100 50 10
Sac2 100 10 100 10
Sal1 10 10 10 100
Sau3A1 100 100 50 10
Sca1 NR NR NR 100
SexA1 100 100 75 50
Spe1 100 75 100 25
Sph1 100 100 100 50
SphI
SSpI
Sty1 10 10 25 100
Sty1-HF 100 25 100 25
Xba1 100 10 100 75
Xcm1 100 10 100 25
Xho1 100 75 100 100
Xma1 100 25 50 10

[^1] gone as of 9/16

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2017S Restriction list

Enzyme Cut Smart NEB 1 NEB 2 NEB 3 NEB 4
Aat2 100 10 50 50
Acc651 25 10 75 100
Aci1 100 10 25 100
Acl1 100 10 10 10
AflII 100 50 100 10
Age1 75 100 75 25
AlwN1 100 10 100 50
Apal1 100 100 100 10
Asc1 100 10 10 10
Ava1 100 10 100 25
Ava2 100 50 75 10
BamHI-HF 100 100 50 10
BciVI 100 100 25 10
Bgl2 10 10 10 100
BmgB1 10 10 10 100
BmsAI 100 50 100 100
BsaI 100 75 75 100
BspD1 100 25 75 50
BspE1 10 10 10 100
BsrB1 100 50 100 100
BsrG1 25 25 100 100
Cla1 100 10 50 50
Dra1 100 75 75 50
Eag1 10 10 25 100
Ear1 100 50 10 10
EcoR1 50 25 100 50
EcoRV-HF 100 25 100 100
HaeIII 100 50 100 25
Hha1 100 25 100 100
Hind3 50 25 100 50
Hind3 HF 100 10 100 10
HinP1I 100 100 100 100
Hpa1 100 10 75 25
Hpa2 100 100 50 10
Kas1 100 50 100 50
KpnI-HF 100 100 25 10
Mbo1 100 75 100 100
Mlu1 25 10 50 100
Msp1 100 75 100 50
Nae1 100 25 25 10
Nco1 100 100 100 100
Nde1 100 75 100 100
Nhe1-HF 100 100 25 10
Pst1 50 75 75 100
Pvu1 10 10 25 100
Pvu2 100 50 100 100
Rsa1 100 25 50 10
Sac1 100 100 50 10
Sal1 10 10 10 100
Sau3A1 100 100 50 10
Sca1 NR NR NR 100
SexA1 100 100 75 50
Sma1 100 10 10 10
Spe1 100 75 100 25
Sph1 100 100 100 50
SSpI 50 50 100 50
Sty1 10 10 25 100
Sty1-HF 100 25 100 25
Xba1 100 10 100 75
Xcm1 100 10 100 25
Xho1 100 75 100 100

G&GA solutions

Make solutions (Check supplies first)

See stock solution recipes


Aliquot:

Solution vol aliquots/pair total aliquots for Labs
T10E1 1 mL 5 50 1.1, 1.2, 2.4, 3.3
T10E5 0.75 mL 2 20 1.1
EtOH 95% 1.5 mL 2.2 22 1.1, 5.4
EtOH 70% 7.5 mL 1.2 12 1.1
isopropyl 7.5 mL 1.2 12 1.1
NaOAc 100 µL 1.2 12 1.1
KOAc 5 mL 1.2 12 1.1
DEB 10 mL 1.2 12 1.1
Tris pH 7.5 10 mM 1.5 mL 3 30 1.2, 5.4
loading dye 50 µL 1.5 15 1.2, 2.4, 2.5, 3.4, 5.4, 5.6
HMW std 15 µL 1.2 12 1.2
dNTP 2.5 mM 150 µL 1.5 15
sterile H2O 1 mL 6 60 2.4, 2.5, 3.3, 5.5
LMW std 50 µL 1.5 15 2.4, 2.5, 3.4, 5.4, 5.6
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solutions calcs

Forpairs
Reagent vol/aliquot aliquots/pr tot aliquots total vol
T10E1 mL
T10E5 mL
EtOH 95% mL
Isopropyl mL
NaOAc mL
KOAc mL
Loading dye mL
Mass ruler mix mL
dNTP 2.5 mM mL
Sterile water mL
Tris pH 7.5 10 mM mL

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G&GA stations

Each Station should have the following items:

Top Drawer

  • 2 gel rigs
  • 2 gel trays
  • 2 pencils
  • 1 alcohol-resistant marker, not Sharpie
  • 1 scissors
  • 1 forceps
  • 1 roll label tape
  • 3 micropipettors: 20μL, 200μL, 1000μL
  • 3 boxes pipet tips: 20μL, 200μL, 1000μL
  • spatula
  • funnel
  • 1 package 10 mL pipettes
  • 1 10mL pipet filler
  • 2 microfuge racks
  • 2 test-tube racks
  • scotch tape
  • ruler
  • timer

Second Drawer

  • 2 250 mL flasks
  • 1 1L beaker of microfuge tubes
  • 1 freezer box

Third Drawer

  • 2 gel casting trays
  • 2 levels
  • 2 15-well combs
  • 2 8-well combs

Each bench should have the following to be shared:

  • 1 waste beaker
  • ice bucket
  • 1 box Kimwipes
  • microfuge
  • DNA Engine Thermocycler
  • Dissecting microscope
  • Parafilm
  • 3 boxes gloves: sm, med, lg
  • Biology of Plants by Peter Raven

Planting guide

Check seed stocks before planting - some germinate better than others!!

Hydroponics video: https://www.youtube.com/watch?v=c9neVLaS63c

Hydroponics paper: http://www.plantmethods.com/content/9/1/4

2012 Schedule

Potting on soil

date wks before Lab purpose per pot pots/pair # pots
1/4 8 0.3 demo 3-5 1 10
1/11 7 0.3 demo 3-5 1 10
3 1.1 DNA lots! 4 40
1/18 6 0.3 demo 3-5 1 10
2 1.1 DNA lots! 4 40
1/25 5 0.3 demo 3-5 1 10
2/1 4 0.3 demo 3-5 1 10
2/8 3 0.3 demo 3-5 1 10
2/15 2 0.3 demo 3-5 1 10
2/27 6 5.3 projects 3-5 1 10
3/6 5 5.3 projects 3-5 1 10
3/13 4 5.3 projects 3-5 1 10
3/20 3 5.3 projects 3-5 1 10
3/27 2 5.3 projects 3-5 1 10

Planting on plates

date for Lab purpose # plates notes
2/22 0.3 demo 10 1 weeks before lab
3/26 5.3 projects 10 2 weeks before lab
4/3 5.3 projects 10 1 weeks before lab
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Calendar Spring 2012

January 2012

Sun Mon Tue Wed Thurs Fri Sat
1 Holiday 2 3 4 Plant 5 6 7
8 9 10 11 Plant 12 13 14
15 16 Holiday 17 18 Plant 19 20 21
22 23 24 25 Plant 26 27 28
29 30 31


February 2012

Sun Mon Tue Wed Thurs Fri Sat
1 Plant 2 3 4
5 6 7 8 Plant 9 10 11
12 13 14 15 Plant 16 17 18
19 20 Holiday 21 22 23 24 25
26 27 Plant 28 29


March 2012

Sun Mon Tue Wed Thurs Fri Sat
1 2 3
4 5 6 Plant 7 8 9 10
11 12 13 Plant 14 15 16 17 Spring Break
18 19 20 Plant 21 22 23 24
25 26 27 Plant 28 29 30 31


April 2012

Sun Mon Tue Wed Thurs Fri Sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 Holiday 17 (Mon) 18 19 20 21
22 23 24 25 26 27 28
29 30


May 2012

Sun Mon Tue Wed Thurs Fri Sat
1 Last day 2 3 4 5
6 7 8 9 10 11 12
13 14 15 Grades due 16 17 18 19
20 21 22 23 24 25 26
27 28 Holiday 29 30 31

On plates

Planting on Plates

Make MS plates: http://wahoo.nsm.umass.edu/content/ms-plates

  • To get weakling mutants started before potting in soil
  • Or to plant seeds on specified media
  • Or to see roots

Seed Preparation

  • Put appropriate number of seeds in a sterile microfuge tube
  • Sterilize with EtOH (or sterilize with bleach – see below)
  • Sterilize filter paper with EtOH, let dry in sterile hood (can set it on top of an open sterile petri plate cover
  • Add 95% EtOH to seeds for 5 min, dump seeds onto filter paper
  • let dry, tap onto agar
  • Make a line of seeds in agarose above the midline of the plate:
  • Tape plates with Micropore surgical tape
  • Refrigerate the plates for 2 days
  • Move plates to growth chamber
  • Stand plates on edge under the lights. Tape several together.

OR Sterilize with bleach solution

  • Add ~1mL of bleach solution to each tube
  • Cap and shake
  • Leave tube on its side for 20-30 min, shaking periodically
  • Put upright after last shake (so seeds settle)
  • In laminar flow hood
    • Use a pipetman that has been sprayed with ethanol and air-dried in the hood
    • Remove as much bleach solution as possible from the tube
    • Add ~1 mL sterile ddH2O, mix and let seeds settle again
    • Do at least 4 washes, to dilute out the bleach
    • Add ~1mL sterile 0.1% agarose (so seeds will be suspended in the liquid)

Bleach solution for sterilizing seeds

  • 7 mL sterile ddH2O (must be freshly made!)
  • 3 mL Chlorox
  • 1 uL Triton X-100 (detergent to help get bleach into seed crevices)

0.1% agarose for plating seeds

  • 100 mL sterile ddH2O
  • 0.1 g agarose . autoclave to melt; shake bottle after autoclaving

On soil

Arabidopsis planting guide - Plants on soil

Routine weekly planting to produce plant material for observation or DNA extraction

  • Pots
    • 6 pots in a big flat with no holes
    • fill with clump-free potting soil, leaving some space at the rim
  • Treat with Gnatrol for fungus gnats
    • saturate with hot water the night before Teddi comes
    • Arrange for Teddi to do Gnatrol treatment
  • OR Heat in oven
    • pre-heat oven to 225F (=~107C)
    • Put soil-filled pots in aluminum lasagne pan
    • Cover with foil
    • insert thermometer in one pot near center
    • Heat to 180F (=~82C)
    • leave in oven for 30 min.
    • leave covered until ready to plant
  • Seeds for DNA
    • Start a set 4 weeks before needed, another set 3 weeks before
    • Columbia wild type (col-0)
    • Scatter seeds on surface of soil (remoisten before if it is dry)
    • Put on plastic cover
    • Put in refrigerator, keep dark (this synchronizes germination)
    • Transfer to growth chamber on the third day
    • If no room in fridge, put seeds in microfuge tube, add water, refrigerate in dark 2 days, then plant
  • Seeds for demo or experimental plants
    • Put seeds in microfuge tube, water, refrigerate, keep dark
    • 3rd day: pour into small beaker, add more water
    • Deposit one seed to each corner of the pot, plus one in the middle with a transfer pipet.
    • Transfer to growth chamber
  • Growth Chamber
    • 22C, 16H light, schedule continuous program
    • Water bottom flat ~3x per week
    • Remove cover after plants are well established.

hydroponics

http://www.plantmethods.com/content/9/1/4

http://www.bio-world.com/productinfo/3_44_292/124429/Magenta-GA-Plant-Cu...

2015 attempt

  • Make 1x MS, pH to 5.6

  • Split: most as growth medium, a little bit for 0.7% agar (err on the low end), sterilize

  • Cut lids off microfuge tubes, poke a hole in each, sterilize in a beaker

  • line lids up, flat side down, on a strip of scotch tape

  • fill with 0.7% agar.

  • put lids into floatie or other rack.

  • put 1 sterilized seed in each hole. (in 0.1% agar)

  • Wrap with saran wrap

  • Cover tightly with foil, stratify in refrigerator for 2-3 days

  • Remove foil, put container in growth chamber at 16h day, 22C

  • Transfer lid to 50 mL tube with hole drilled in it at ~3 weeks.

  • Aerate or stir medium

cDNA

*

cDNA 2010

*Making cDNA +Fe & -Fe roots and shoots**

20 MS plates, heavily sown with At Col0 seeds.

At 7 days, transplant all seedlings, half to MS plates, half to –Fe plates.

After 3 more days, harvest roots, shoots. 12 samples in all.

(In future, plant 4 full plates per root sample)

Use razor blade to amputate roots

Grind in ceramic mortar and pestle (bake 1st overnight!)

Extract RNA, following lab manual protocol.

From 5/7/10:

Sample ng RNA/uL uL to get 200 ng uL to get 44.5 ng H2O to 5 uL
R+1 23.84 8.39 1.87 3.13
R+2 95 2.11 0.47 4.53
R+3 24.5 8.16 1.82 3.18
S+1 67.35 2.97 0.66 4.34
S+2 32 6.25 1.39 3.61
S+3 73.6 2.72 0.60 4.40
R-1 9.5 21.05 4.68 0.32
R-2 8.9 22.47 5.00 0.00
R-3 18 11.11 2.47 2.53
S-1 98.75 2.03 0.45 4.55
S-2 76.45 2.62 0.58 4.42
S-3 79.05 2.53 0.56 4.44

max ng in most dilute sample= 44.50

Can’t get 200 ng RNA in less than 5 µL in several samples.

Calculate the amount of RNA in 5 µL of the most dilute sample (-R2), calculate the µL of each sample required to get that amount. Add water to 5 µL.

Run on gel. R-3 was calculated wrong (or it degraded).

Initial spec reading was 6.3; reblanked and got 18. Maybe the 6.3 was right?

Gel:

Lane 1 2 3 4 5 6 7 8 9 10 11 12 13
HMW std R+1 R+2 R+3 S+1 S+2 S+3 R-1 R-2 R-3 S-1 S-2 S-3

Freeze the RNA.

Two choices for cDNA:
* omit R-3 (calculate amount of RNA from next most dilute, -R 2), * or recalculate R-3 from the initial 6.3 reading

From –R2: 11 µL contains 97.9 ng of RNA, almost 10% of the recommended amount.

Can run 11 RTPCR reactions, and only have 40 µL of R- samples, but 60 µL of all of the others.

From –R3 at 6.3ng/µL: 11 µL contains 69.3 ng, which is only 7% of the recommended amount.

Combine the 3 of each type into a single tube. Measure volume:

ingredient R- R+ S- S+
uL RNA 102 111 111 111
+ 1/10 vol 3M NaOAc 1 10.2 11.1 11.1 11.1
+ 2 vol EtOH 204 222 222 222

Freeze 20 min

Spin 10 min

Remove supernate

Add 70% EtOH

Spin again (R pellets hard to see)

Remove all liquid. Add:

ingredient R- R+ S- S+
1/4 vol RNAse-free H2O 25.5 27.75 27.75 27.75
spec ng/uL: 118.2 116.5 282.4 203

11 µL of least conc has 1281.5 ng

ingredient R- R+ S- S+
uL RNA 10.84 11 4.53 6.31
water 0.16 0 6.49 4.69

Proceed with RT, following lab manual protocol.

Run gel (1st one failed – pic is second run)

LMW 1/50 1/100 R+ S+ R- S- std gDNA gDNA

Complete waste of time and resources!

Test the cDNA with PCR

Test cDNA with oMZG_RT_L and oMZG_RT_R

(The primer names in the Wiki are oMZG_T_R and oMZG_T_L; in the freezer, there is oMZG_RT_L and oMZG_RT_R)

gDNA product is 482bp, cDNA is 362 (with some alternative splicing products in between).

Master Mix for 7 rxns (1/50 gDNA, 1/100 gDNA, R+, S+, R-, S-) + 1

ingredient per rxn per 7 rxns

water 36 252

10X buffer 5 35

2.5mM dNTP 4 28

oMZG_RT_L 1 7

oMZG_RT_R 1 7 T aq 1 7

Each rxn: 48 µL master mix + 2 µL template

Make 1/50 gDNA: 1 µL gDNA + 49 µL water

Make 1/100 gDNA: 10 µL 1/50 DNA + 10 µL water

Tube # 1 2 3 4 5 6 Sample 1/50 gDNA 1/100 gDNA R+ S+ R- S-


  1. make fresh, pH 7.0, filter sterilize 

cDNA 2013

Make cDNA Summer 2013

Summary:

12 samples, 3 replicates of each treatment:

MS -> MS MS -> -Fe
shoots numbers 1-3 numbers 7-9
roots numbers 4-6 numbers 10-12

cDNA in 12 labeled tubes in freezer in 362 (each made with 1 µg RNA, using superscript III)

RNA in box in -80 in 262

number plant part Fe ng/µL µL RNA/1µg
1 shoot Fe+ 125.9 7.94
2 shoot Fe+ 328.5 3.04
3 shoot Fe+ 259.2 3.86
4 root Fe+ 318.1 3.14
5 root Fe+ 429.5 2.33
6 root Fe+ 282.6 3.54
7 shoot Fe- 883.7 1.13
8 shoot Fe- 427.9 2.34
9 shoot Fe- 432.4 2.31
10 root Fe- 224.3 4.46
11 root Fe- 258.8 3.86
12 root Fe- 139.9 7.15

sterilize seeds

  • sterilize seeds in 0.5 mL 70% EtOH, 0.05% TritonX-100, ~3 min, mild agitation.

  • Let seeds settle, remove liquid

  • Replace with sterile water, 3X

  • Replace water with sterile 0.1% agar, 3X

  • plate heavily in a line on flattened cellophane (wet it if necessary)

  • stack plates flat

  • cover in foil, refrigerate, 48 hours

test platforms

Try different porous platforms for seedling growth

Get samples from Magdalena's lab

Sterilize between sheets of filter paper in a glass petri dish.

  • Moss cellophane

  • Roll cellophane

  • Magenta box screen

  • Sheer fabric

Seeds grow fine on flat cellophane. The roots have trouble getting over a bump or air bubble.

Seeds do not grow well on mesh fabric.

Wire screen does not lie flat on agar.

Put samples of each on MS agar

Iron experiment

Grow Col0 seeds to test effect of iron deprivation on gene expression

Make plates:

Sow sterile seeds on cellophane in 12 MS plates as heavily as possible.

After stratification,

  • 7 days in incubator

  • Transfer half to iron-free plates, half to MS plates

  • Harvest after 3 days

Extract RNA

Grind tissue

Grind roots +/-, shoots +/- in ball mill (Sam's Retch Mixer Mill MM400)

  • Precool the white block for the ball mill

  • prepare 2 2-mL tubes per plate (label tubes on side as well as top)

    • 3 roots + iron
    • 3 roots - iron
    • 3 shoots + iron
    • 3 shoots - iron
  • 2 metal beads per tube 3.2mm diameter ss beads (90g) - Cat. No. 11079132ss from www.biospec.com (Can also get from Fisher)

  • cool in LN2

  • Add tissue to cold tube, put back in LN2

  • Put all tubes in cold block

  • Shake 20 sec, check. The heat of shaking may start to defrost the tissue. Throw it back in the LN2. (Shake too long, and the cap may disintegrate!

  • Return to LN2

RNEasy

Prepare RNA from powdered frozen tissue

Use RNeasy Plant Mini Kit (50) Qiagen 74904

  1. Add 450 µL Buffer RLT. Mix vigorously. Spin down.

  2. Transfer lysate to lilac Qiashredder in 2mL collection tube

  3. Spin 2 min, full speed

  4. Transfer flow-through (NOT PELLET) to new tube. Toss the lilac column

  5. Add 225 µL 100% EtOH to lysate. Mix by pipetting up and down

  6. Transfer entire sample to pink spin column in a 2 mL collection tube.

  7. Discard flow-through. Keep column.

  8. Add 350 µL RW1 to column. Spin 15 sec, full speed.

  9. Discard flow-through. Keep column

  10. Add 70µL RDD to 10 µL DNAse I. BE GENTLE. Put the 80 µL on the column. Incubate at room temp 15 min.

  11. Add 350 µL RW1. Spin 15 sec. full speed.

  12. Discard flow-through, reuse collection tube.

  13. Add 500 µL RPE to column. spin full speed 2 min.

  14. Put column in new capless collection tube. Discard old collection tube.

  15. Spin 1 min, full speed

  16. Add 30 µL RNAse-free water to column membrane. Spin 1 min, full speed.

  17. Repeat, with 20 µL RNAse free water.

  18. Transfer all 50 µL RNA to clean, labeled tube. Keep on ice

Reagents

For RNEasy reactions
add: mL RLT
add: mL 100% EtOH
add: mL RW1
add: mL RDD
add: mL DNAse I
add: mL RPE
add: mL RNAse-free water

[RNA] by nano-drop

  1. Clean the pedestals
  2. Blank the machine

    1 µL buffer onto lower measurement pedestal

    F3 (Blank)

  3. Wipe again
  4. Test the blank:

    1 µL buffer

    F1 (Measure)

    if it's flat, you're good to go

  5. 1 µL sample. repeat.

RT

Make cDNA by reverse transcription

Calculate the volume of each sample required to get 1 µg RNA

For reactions
with ng/µL RNA
add: µL oligo(dT)
add: µL RNA
add: µL 10mM dNTP mix
add µL water (final vol = 13µL/rxn) 65 C 1 min, then ice. Spin down.
add: µL 5X 1st strand buffer
add: µL 0.1M DTT
add: µL RNAseOUT
add: µL Superscript III (200U/µL). 50C 45 min. Stop rxn: 70C 15 min

Note I bought the 10 µL Superscript, mixed it with some leftover, and had enough to do one reaction per RNA sample (=60 µL per treatment). Should have bought the bigger size.

I should use some of the remaining RNA to try out the RT sample I got from Qiagen. Maybe the best 8 RNA's.

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