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-