December 8th + new findings from a paper found

On December 1st we saw issues with the amount of RT-PCR product we were loading into our gel. On December 8th we decided to re-run the gel using the stored product. We assumed that each sample was around 15ul and added 1.5ul of 10X loading dye to each tube. We added 10ul to all wells and found no results the second time as well.

Upon further research a paper by Masood Siddique et al. published in Cell Stress and Chaperones (2008) “The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties” elucidated as to why we might have gotten no data.

What we learned from the paper: HSP18.5 is not expressed under Salt stress, it is only active during Heat stress. Not only this, it has a very low expression rate in root tissue, but is much more active in shoots and leaves. With the HSP superfamily being active in many different stresses it was tough to find which specific HSP would be related to salt stress, and we picked the wrong one.

This finding would have saved us precious time and we could have chosen a different primer set for our RT-PCR.


HAC1/Ubq normalization

Hey Everyone, sorry this is so late. Here is the info for the HAC1 expression normalization to Ubiquitin expression. We quantified expression levels of HAC1 and Ubq from gel images using the imageJ software (calulate total area pixels in gel, find percentage of that total that came from each lane in the gel, and set it to a standard). Also, DREB2B is our specific gene so you probably don’t need to worry about that column. Hope this helps.

WT3(root) 1.00 1.00
WT6 (leaf) 1.52 0.02
WT6 4.32 6.18
WT7 4.90 0.86
WT8 3.17 0.87
WT10 17.97 2.42
WT12 12.30 1.10
WT13 – gDNA contaminated? 0.00 0.00
WT14 1.74 1.34
WT15 1.23 1.39
M2 3.93 0.10
M5 0.94 0.03
M6 0.00 0.00
M10 0.00 0.00
M13 14.21 2.09
M18 4.99 0.14
H20 21.37 2.63
No RT 24.33 3.67


AC green troubleshooting UBQ primers


1 Laddder – not loaded

2 gDNA old primers

3 H2O New UBQ10
4 gDNA (WT2) New UBQ10
5 cDNA (WT3) New UBQ10
6 cDNA (WT6) New UBQ10
7 H2O UBQ5
8 gDNA (WT2) UBQ5 (only 7-8ul)
9 cDNA (WT3) UBQ5
10 cDNA (WT6) UBQ5 (only 7-8ul)

AC Green Journal club artricle

We chose to do our journal club article on Tomato Heat Stress Transcription Factor HsfB1. The article provides evidence  that HsfB1 represents a novel type of coactivator cooperating with class A HSFs. The article also provides evidence that HAC1 acts as a transcription factor, which is a finding that our group thought could help us with future studies with HAC1 and our current study with HAC1.

AC green troubleshooting

purpose: to trouble shoot our UBQ primers and assess the quality and binding capabilities of old primers compared to new ones (UBQ 5 and UBQ 10). We wanted to see if the old spring 2010 UBQ primer had actually degraded and if that was the reason why we were getting negative results for our RT-PCR.


gDNA wt2 (our sample)

cDNA treated with salt from F10ACw6

cDNA untreated F10ACW3

H20 control

we loaded 1 microliter of each sample onto our gel and tested gDNA with our old UBQ10 primer, new UBQ 10 primer (which we designed) and new UBQ5 primer (from Liu et al literature). We also tested both cDNA samples with new UBQ10 and new UBQ5 primers.


The original primers we used did show non-specific biding and degradations of gDNA. New UBQ10/5 target both cDNA and gDNA and are strong in recognizing ubq in cDNA. UBQ5 also shows binding to cDNA. Both would be good alternatives to use for normalization. We saw different sized bands on the gel because each set of primers has a different product size.

AA Green Troubleshooting Experiment

For our troubleshooting experiment we were assigned the task of attempting to germinate A. thaliana on MS agar medium.  We researched several publications and came to on consensus as a group as to what concentration of MS to use and what percentage of agar to use as well.  Here is the protocol we came up with:

Germination of Seeds on Agar (MS Media) Protocol:

0.5 MS Media with 0.1% Agar and 2% Sucrose

1. Added 2.16 g MS Salts to 0.9 L of distilled H2O and let dissolve.

2. Measure pH.  The pH should be around 5.7.  Adjust if necessary.

3. Diluted to final volume of 1L and added agar (10g/L0

4. Autoclaved

5. Added sucrose after media cooled before solution was poured in petri dishes. (20g Sucrose)

6. Sucrose dissolve(swirled the solution) before pouring into the petri dishes.

7. Poured approximately 85 mL of the solution into each petri dish.

8. MS media solidified overnight (in the hood)


Sterilization of Seeds

-15mL of Bleach + 15 mL dH2O = 0.5Bleach

-Added 15 microliters of Tween 20 to Bleach (0.05% Tween 20)

1. Washed Wild Type seeds with 0.5 Bleach, and then it was poured off.

2. Washed 4x with 10 mL sterile H2O (inverted 10-20 times each time)

3. Poured off water after seeds settled to the bottom

-Used 1000L pipet to suck up remaining water

4. Added 2 mL sterile H20 each time the seeds were plated

-Plate 1 –> 400 microliters H2O + seed on plate + 1 mL H2O onto plate

-Plate 2 –> extracted 300 microliters, added 2 mL H2O to tube, then distributed 1.7 mL onto plate

-Plate 3 & 4 –> Resuspend seeds & extract 2mL of H2O + seeds & distribute on plate

5. Tilted plates slightly and removed excess H2O from plate sides using widetip L200

Plate 2:

-add water to make 2mL H2O into tube

-remove 300microLiters of seed + water and distribute onto plate

-add water to make complete 2mL H2O level in tube

-remove and distribute remaining 1.7L H2O of water + seeds onto plate

We found that adding 2mL of H2O made for an easier distribution of seeds on the plate.  We did have some trouble with light issues though and that may have had an adverse effect on plant growth.  The lights weren’t on for 16 hours like they should of been.  Pictures of the plate with seeds that were germinated on it can be seen in the class photos section.  We also found it very easy to remove the plants intact from the agar (including roots) after they had germinated.  Pictures of this can also be seen in the class photos section.


AC_Blue_cDNA synthesis, Thursday (12/01/11)

On Thursday, Dec 1st  the blue AC group prepared 16 cDNA samples and ran one PCR for our hsp18.5 primers in the order indicated by Professor Crowe and Peter ( image below). 




Standard protocols for PCR were followed as indicated on page 34 of our lab manual.  Unfortunately, after running PCR and exposing the gel to UV light, we did not see any bands (shown below).  We were only able to see the top DNA ladder, but including our gDNA, leaf or root wild-type controls did not appear on the gel.  We assume that there might have been inhibition between our products or that we didn’t have all the elements needed for the reaction in our “master mix”.