cDNA

cDNA kdorfman Mon, 01/16/2012 - 16:22

*

cDNA 2010

cDNA 2010 kdorfman Fri, 05/24/2013 - 16:45

*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

cDNA 2013 kdorfman Fri, 05/24/2013 - 16:45

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 kdorfman Fri, 05/24/2013 - 18:33

  • 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

test platforms kdorfman Fri, 05/24/2013 - 18:38

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

Iron experiment kdorfman Fri, 05/24/2013 - 18:46

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

Extract RNA kdorfman Fri, 05/24/2013 - 18:49

Grind tissue

Grind tissue kdorfman Fri, 05/24/2013 - 19:13

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

RNEasy kdorfman Fri, 05/24/2013 - 19:30

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

Reagents kdorfman Mon, 05/27/2013 - 18:15
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

[RNA] by nano-drop kdorfman Fri, 05/24/2013 - 19:00
  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

RT kdorfman Fri, 05/24/2013 - 18:50

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.

rnagelsfeexpt.xls
rnamass.xls
rna-for-fe-exp.jpeg
rna-for-fe-exp2.jpeg