*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.
|Sample||ng RNA/uL||uL to get 200 ng||uL to get 44.5 ng||H2O to 5 uL|
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?
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:
|+ 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
Add 70% EtOH
Spin again (R pellets hard to see)
Remove all liquid. Add:
|1/4 vol RNAse-free H2O||25.5||27.75||27.75||27.75|
11 µL of least conc has 1281.5 ng
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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