*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-
-
make fresh, pH 7.0, filter sterilize ↩︎
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