AtGRP7 analysis

Using data from the 5hpi, 7hpi and 12hpi to make a new excel file.



Using the mature as the reference:

qPCR - 12hpi and UPF PR-1 stuff

Started qPCR for the UPF PR-1 stuff, as well as the AtGRP7 stuff for the 12hpi, since I forgot to add some primers last time.

2ul cDNA for each mastermix.

Everything: .pcrd        PDF
UPF PDF .xls
AtGRP7 PDF .xls

UPF PR-1:

Looks like PR-1 isn’t really induced in the UPF mutants. I’m not sure why I saw something in the Col-0 though.

AtGRP7 with Mature as reference and At3g25470 as reference:


Combining the data from this run and the last run for 5 & 7 hpi to make a new master file. Analysis here.

AtGRP7 qPCR #2

Setup the plate for qPCR of the previous samples, AtGRP7 stuff only.

Mastermix, one for each of 9 samples:
105 2x Bio-rad Sybr
18 water
3 cDNA

Primer mastermix:
66 primer (At3g25470, AtGRP7_spliced, AtGRP7_mature)
198 water

*** Just realized I didn’t add the mature splice primers for the 12hpi hrcC sample. Damn it! ***

.pcrd                PDF

All:


5hpi, 7hpi, 12hpi:


5hpi, 7hpi, 12hpi using the mock as a control:


5hpi and 7hpi using mock as control and Mature as reference (instead of At3g25470)


At first glance, everything is the same as last time we used mature as the reference, except for the 7hpi hrcC. This time shows a drop in the spliced versus the mature at 7hpi, but before it didn’t drop at all, and stayed about a 1:1 ratio the entire time. Last time’s 5hpi and 7hpi (mock isn’t shown, but is set to 1):


Have to re-do the 12hpi stuff... found here

RNA isolation, day 2 - bioanalyzer, cDNA prep

Submitted the RNA to bioanalyzer. I used 2ul DEPC water and 2ul of the RNA for dilutions.

Order # 1191

Everything looks good.



12/23/10 12:39 PM
Started cDNA using the iScript reaction kit.

3ul RNA
4ul 5x mix
1ul reverse transcriptase
12 water (from the kit)



qPCRs are going to be in there own separate entries:
AtGRP7 5&7hpi        12hpi
UPF

RNA isolation

Isolating RNA from the Col-0 infiltrations yesterday for the AtGRP7 stuff. Also getting the UPF RNA without any treatment, just to see if PR-1 is constitutively on compared to Col-0.

Samples:
1        Col-0
2        UPF1-4
3        UPF1-5
4        Mock 5hpi
5        P1 5hpi
6        P5 5hpi
7        Mock 7hpi
8        P1 7hpi
9        P5 7hpi
10        Mock 12hpi
11        P1 12hpi
12        P5 12hpi

3 chloroform washes. Ended up with 500ul of sample, added 500ul isopropanol and put in the -20.

12/22/10 12:17 PM
Spinning for 10 minutes at room temperature.

12/22/10 1:01 PM
Made mastermix for DNAse:
1 glycogen
3 10x DNAse buffer
26 DEPC H2O

Resuspended the pellets in 29ul of this mastermix, and then added 1ul DNAse. 37* for 10 minutes, followed by 70* for 10 minutes. Added 75 100% EtOH, then -20*.

12/22/10 4:05 PM
Spinning for 10 minutes at room temperature.

40ul resuspension in DEPC water.

Sample ID	ng/ul	260/280	260/230
Col-0	322.73	2.09	2.29
UPF1-4	230.38	2.09	2.27
UPF1-5	264.25	2.1	2.29
Mock 5hpi	368.82	2.09	2.25
P1 5hpi	415.91	2.09	2.25
P5 5hpi	447.06	2.1	2.27
Mock 7hpi	324.73	2.09	2.27
P1 7hpi	231.85	2.1	2.27
P5 7hpi	380.32	2.1	2.26
Mock 12hpi	375.76	2.08	2.23
P1 12hpi	431.2	2.09	2.26
P5 12hpi	305.23	2.09	2.21

The CGRB website isn’t working, so I will submit this for bioanalyzer tomorrow.

Col-0 infiltrations 5, 7, 12 hpi for RNA

Yesterday I started cultures of P1 and P5 for infiltrations. Spinning down today.

Dilutions

NOT doing additional cyclohexamide. I’ll save that for the dipping method.

Infiltrating into Col-0 from 2010-10-18. I did 18 leaves with 10mM MgCl2, P1 and P5. I am putting the plants in the long day, since I have to do a 12hpi timepoint. This way, the plants will be in the light the whole time, and there will hopefully be nothing weird going on with the AtGRP7 circadian rhythms.



Take samples at 2:30, 4:30 and 9:30.

2:30 sample
The leaves were pretty big, so I only took 3 from each.

4:30
Everything looked good, 3 leaves each.

9:30
Everything looked good, 3 leaves each.

Setting up for re-do

Started cultures to do the 5, 7 and 12hpi experiment for tomorrow.