Amanda Tabbert
From Shiu Lab
My project will be testing Arabidopsis thaliana and Arabidopsis lyrata under different stress conditions including cold/freezing tolerance, salt (continuous and transfer), drought (dehydration for seedlings and adults), and lastly osmotic (PEG and Mannitol). I will not only look at the difference between these two species but I am going to preform the stress tests on different ecotypes as well. Eventually, I would like to be able to identify which genes are being expressed under the varying stress conditions.
Lab Notes
June 2007
June 20, 2007 A. lyrata seeds sterilized and in fridge (4C) 1) A. lyrata sequenced strain (22696) 2) A. lyrata Oval Beach at Kalamazoo Road 3) A. lyrata NE Saugatuck #1 4) A. lyrata NE Saugatuck #4 5) A. lryata NE Saugatuck #5. Made Agar MS + Sucrose and plated
June 22, 2007 Planted A. lyrata on MS + Sucrose and place in fridge (4C) 1) A. lyrata sequenced strain (22696)
2) A. lyrata Oval Beach at Kalamazoo Road 3) A. lyrata NE Saugatuck #1 4) A. lyrata NE Saugatuck #4 5) A. lryata NE Saugatuck #5 June 27, 2007 Seed plates moved from fridge (4C) to growth chamber (21C) -except one plate of 22696 which will be kept in the fridge for long vernilization to see if that will increase time to bolt and flower.
July 2007
Monday, July 09, 2007 1. Transferred A.lyrata seedlings from MS + Sucrose to soil treated with gnatrol. a. NE#4 Saugatuck 9/13 germinated and planted b. NE# 1 Saugatuck 5/6 germinated and planted c. Oval Beach at Kalamazoo Rd 2/5 germinated and planted (3 still on plate to finish opening cods) d. Sequenced strain 10/10 germinated and planted e. NE#5 Saugatuck left to grow on plates a little longer (cods not fully expanded) Wednesday, July 11, 2007 1. Transferred A.lyrata seedling from MS + Sucrose to soil treated with gnatrol. a. NE#5 Saugatuck 4/5 germinated and planted b. Oval beach at Kalamazoo Rd 2/3 remaining (4/5 total) germinated and planted 2. Transferred A.thaliana Columbia seedlings to soil w/ gnatrol from MS + Sucrose.(18 total transferred)
Friday, July 20, 2007 Plants NE#5-3, NE#1-1, and Oval Beach-1 died and taken out of growth chamber. Survival impaired by transferring to soil too soon?
I have also started to learn from Juyeon about the RNA extraction experiments for Cheng. As she is leaving at the end of July, I will be taking over and continuing her work.
August 2007
8-2-07 Made ms + sucrose plates. Sterilized col seeds for plating (1 mL 100% bleach 5 minutes)- plated them with 1% agar sol’n. 7 plates ready to experiment with on Aug. 16th.
8-6-07 Plated lyrata and col seeds (5 seeds per plate. 3 plates) for Melissa to do experiments with; in the fridge, ready to transfer on Aug. 13. Collected seeds from transformed col and lyrata (ddf1 & 2 RNAi)- drying in Melissa’s cupboard.
8-8-07 Collected seed from the rest of Cheng’s Lyrata and Columbia.
8-10-07 Plants 3, 5, 6 from Lyrata sequenced strain died due to mildew on them. The rest look great except the plants we received from the stock center. I also took the covers off of Cheng’s Columbia seedlings she will use for transformants.
8-13-07 Sterilized and planted Melissa’s transformed lines (50% bleach 3min). 1) DDF1 PEG 203 A 2-15-07 3-27-07 2) DDF2 PEG 203 B 2-15-07 3-27-07 3) DDF1 PEG 203 B 2-2-07 3-27-07 4) DDF2 PEG 203 B 2-2-07 3-27-07 5) DDF1 PEG 203 A 7-18-07 or 7-13-07 6) DDF2 PEG 203 A 7-18-07 or 7-13-07 For # 5 & 6 I couldn’t tell if it was 13 or 18. Sterilized seeds and put in fridge (100% bleach 3min) 1) DDF1-4 pMDC 163 A 7-13-07 2) DDF1-1 pMDC 163 B 7-13-07 3) DDF2-2 pMDC 136 A 7-13-07
8-27-07 Moved lyrata seeds from fridge (long vernilization) to 24 hr light. They were in 4C for 8 weeks. 2 seeds germinated on the plate. Sterilized seeds for hyg selection 100% bleach for 3 minutes; in fridge until Friday. 1) DDF2-2 pMDC163 B 2) DDF1-4 pMDC163 C 3) DDF1-1 pMDC163 B A. lyrata sequenced plant #7 died- gnats ate
September 2007
9-5-07 Plated the rest of melissa’s DDF1-4 pMDC 163 B on hyg + MS + 1% sucrose plates. I got 2 plates of DDF1-1 pMDC 163 C on hyg + MS + 1% sucrose done. Made more HYG + MS plates no sucrose.
Transferred one A. Lyrata seedling to soil from the 2 month vernilization in 4C, doesn’t look like any more will germinate.
9-12-07 Long vernilization seeds did not survive. Some germinated but died before large enough to transfer. The one seedling I transferred to soil also died; I don’t know that caused it.
9-17-07 Plated more seeds for hyg selection; haven’t finished stock I’ve been given will work on it all week. Once we get a timer my A. Lyrata (and some of cheng’s) are going into a cold room to make them flower.
9-24-07 DNA preps for Melissa
9-28-07 Plated putative transformants for Melissa last week and now transferring them to soil. Made 50xTAE and prepared and ran PCR with the DNA preps from Monday.
October 2007
10-3-07 DNA preps on Melissa’s plants #1, 3, 6, 8, 10, 13 in addition to DDF2 in peg203 1:100, H2O, Agri51/DDF2R, and DDF2F/DDF2R.
10-05-07 PCR on preps
10-08-07 Ran preps on gel
10-16-07 Collected leaves from all of Melissa’s plants for DNA preps (placed in -80 freezer). 1- ddf2 peg 203 b 2- ddf2 peg 203 b 3-ddf1 peg 203 b 4- ddf1 peg 203 b 5-ddf1 peg 203 a 6- ddf2 peg 203 a 7- ddf2 peg 203 a 8- ddf2 peg 203 a 9- ddf2 peg 203 a 10- ddf2 peg 203 a 11- ddf2 peg 203 a 12- ddf2 peg 203 a 13- ddf2 peg 203 a 14- ddf1 peg 203 c3
10-19-07 All my A.lyrata except Oval beach 2, NE #5-1,2, and NE #4-6 which are bolting were moved into the cold chamber for one month vernilization.
10-22-07 Processed DNA preps and in fridge. NE #5 plant 1 and Oval Beach 2 bolted
10-24-07 Ran PCR on preps, ran on gel
November 2007
Nov.5-9, 2007 I was out of the lab and Melissa ran my PCR products on a gel and we have transformants! Numbers 2 and 10 are DDF2 transgenics while 14 is a DDF1transgenic.
November 16, 2007 I am cleaning out the 2nd growth chamber and when all the plants are done drying down we are going to sterilize both growth chambers in hopes if killing any remaining thrips.
November 28, 2007 Last of the plants have been collected and next week we are bleaching and cleaning all 3 growth chambers. From my plants: NE#5-1 and 2; NE#4-6; and Oval beach Kzoo- 2 have died. Too many bugs.
December 2007
All this month I have been selecting RNAi lines on hygromycin plates. So far we have some putative transgenic plants from 5-1 and PHG12 strains. We have transferred them to MS + sucrose plates and then moved them to soil to mature. We will then isolate DNA or perform GUS staining on them to confirm transformation. This selection will continue until I have gone through all the seed for the RNAi lines in Thaliana.
January 2008
1-2-08 To 1-12-08 plated putative transformants on HYG plates for Melissa. I wrapped them in aluminum foil (no light) and placed them in her drawer.
1-14-08 Transplanted putative transformants from plates to soil for Melissa (13 flats). Treated two flats w/ Basta for selection ( DDF1 PEG 203C and PEG 203A).
1-18-08 Collected leaves from DDF2 RNAi pHG12 for DNA prep.
1-23-08 Preformed DNA preps as per protocol. Some lyrata bolted- OB 3, 4; NE#1- 4,5; NE# 4- 2, 7, 8; NE#5-4; Seq. 4, 9.
1-24-08 Ran PCR DDF1 F&R, HYG F&R on DDF2 RNAi pHG12.
1-25-08 Ran PCR products on 1% gel. Something major is wrong. All plants should have a band in DDF1 F&R and they don’t.
1-28-08 Redo PCR with 2, 7, 13, 17, 20, water, + control(DDF1 pMDC83), and Columbia. Used only 25 microliters total reaction volume and MStandard PCR run instead of 50 microliters total and Amanda’s PCR. Planted 79 seedlings for Melissa using 28 cell flats and placed in short-day chamber (not enough room in long-day).
February 2008
2-4-08 Jessica and Melissa figured out the problem with the DNA preps and the PCR (Jessica forgot to add water to the extraction buffer). Redid preps from new leaves for plants 17-25, Columbia from Inga, Columbia stock, pMDC83, and water (DDF2 RNAi pHG12).
Moved A. lyrata out of cold chamber (4 C) and into growth chamber downstairs (21 C). Oct. 19, 2007 to Feb. 4, 2008 in cold.
2-5-08 Ran PCR using DDF1 F & R and also HYG F & R on 17-25, Columbia from Inga, Columbia stock, pMDC83, and water (DDF2 RNAi pHG12). Made 2 L of LB for Melissa according to her recipe. Treated PEGs with Basta.
2-6-08 Transplanted 5 PEGs (possible transformants). DNA extraction/ PCR to follow at a later date to confirm and I autoclaved biohazard today.
2-11-08 DNA preps 1-16 AtDDF2 RNAi PHG 12 planted 1-2-08; 4 Col from Inga (B & C frozen) (D & E fresh). #3 has 2x amount of Tris 1mM pH 8.0. 1-36 Salk 137015 #18 BC2F2 LD in mine and Jess’s box in 4C
2-12-08 Lab meeting
2-13-08 DNA preps for Salk 137015 #15 BC1F2 LD
2-19-08 Lab meeting- wait on Salk pcr; do 1-16 RNAi Phg 12 with DDF1 F&R and Hyg F&R
2-20-08 collected seeds and wax papered Jessica’s plants
2-21-08 PCR on 1-16 RNAi Phg 12
2-25-08 ran PCR products on 1% gel; wrapped Salk 137062 bulk T4 in wax paper. DDF2 RNAi Phg12 #2,3,5,11,15,16 are positive from PCR- kept and wax papered.
2-26-08 lab meeting
2-27-08 Melissa DNA preps: DDF2 RNAi PHG 12 #1-20 (1-25-08) and DDF2 RNAi 5-1 #1-3. For Cheng DNA preps: Ly DDF1 35s pmdc 141 Top 20 #1-9 and Ly DDF1 35s pmdc 141 Top 10 #1-9.
2-28-08 ran another pcr trial with PHG12 #2, 3, 5, 11, 15, 16. Used ColD, verifying transgenic. Agri51/DDF2R and DDF2F&R used DDF@ pmdc 83 as positive control. Watered plants, restaked overgrown plants.
March 2008
3/10/08 ran Melissa’s PCR prep from 3-5-08 on 1% gel (Reverse and LB)
3/11/08 ran Melissa’s PCR prep from 3-5-08 on 1% gel (DDF1 F/R) Prepared DNA preps from Melissa with Jessica’s help
Col from Inga; DDF2 peg 203 A #10 1-4; DDF1 peg 203 c3 #1-4; Salk 137015 #15xCol BC2F2 1-3; Salk 137015 #18xCol BC3F1 1-3; DDF1 pmdc 83 B2 1-4; DDF1 pmdc 83 B1 1-4
3/12/08 PCR with Agri51/DDF1R and Hyg F/R for Cheng’s Ly DDF1 35s pmdc 141 Top 10 and Ly DDF1 35s pmdc 141 Top 20
3/13/08 Ran Cheng’s PCR products on 1% gel. I also ran Melissa’s DDF2 NAi Phg12 2,3,5,11,15,16 with Agri51/DDF2R and Hyg F/R
3/17/08 to 3/19/08 Bagged plants for Melissa
3/20/08 PCR on Cheng’s Ly DDF2 35s pmdc 141 top 10 (1-9) with new DDF1 reverse primer
3/25/08 Complete PCR for Cheng using the new DDF reverse primers
Ly DDF2 35s pmdc 141 Top 10 (A-N); Ly DDF2 35s pmdc 141 Top 20-10 (1-7, 9, 11, 16); Ly DDF2 35s pmdc 141 Top 10-20 (8, 10, 12-15); Ly DDF1 35s pmdc 141 Top 20 (1-9); Ly DDF1 35s pmdc 141 Top 10-20 (1)
3/26/08 Ran PCR products on gel 1%
3/27/08 Hyg F/R on DDF2 positive control to make sure it works before doing all the preps. Pollinated Cheng’s Lyrata in short day chamber.
3/31/08 Painted lab lunch room
April 2008 4/7/08 Collected seed for Melissa. Positive control for DDF2 worked so I ran PCR on LyDDF2 35s pmdc 141 top 10, top 20-10, and top 10-20
4/8/08 Collected seed for Melissa. Ran PCR products on 1% gel. Redo #4 contamination.
4/9/08 Collected seed.
4/10/08 Redid Ly DDF1 35s pmdc 141 top 10 and Ly DDF2 35s pmdc 141 top20-10 with Hyg F/R.
4/11/08 Ran PCR on 1% gel- all positives including neg. control must redo.
4/14/08 Redid Ly DDF1 35s pmdc 141 top 10 and Ly DDF2 35s pmdc 141 top20-10 with Hyg F/R. Ran Melissa’s PCR for shirt day RNAi a-x with DDF2 F/R and Hyg F/R.
4/21/08 Ran short day and DDF2 PHG 12 1/20.
4/23/08 Redid short day Hyg F/R, collected seed, cleaned out growth chamber Ron.
May 2008 Genotyped all of Melissa’s plants using PCR and gel electrophoresis. I collected seed from the gene specific positives. I also cleaned out any remaining plants so we can clean the growth chambers and get rid of the thrips.
Protocols
Seed Sterilization: 1)100% bleach for 3 minutes with a drop of tween. 2)Shake 3) centrifuge for 4 seconds 4) pipette off supernatant and discard 5)add 1000 micro liters sterile water and repeat steps 2-5 four times or until soap bubble are gone.
Plating seeds: when plating seeds for germination use 0.1% agar solution instead of water. 0.1 gram agar per 100 ml water.
Basta selection: For a 325 micromolar solution add 6.5 mg of Glufosinate ammonium to 100 mL of water.
1% gel: 200mL of 1xTAE buffer for every 2 grams agarose (1% gel); Add stir bar. Microwave for 1.5 minutes, when cooling add ethidium bromide (4 micro liters per 100 mL of gel). When cool to touch, while stirring, pour gel into mold.
Hyg Plates follow MS + Sucrose protocol but leave out sucrose and add 200 microliter HYG per 500 ml of sol'n when it has cooled to the touch after autoclaving (21 minutes liquid cycle); stir w/ magnetic stir bar, pour plates.
50xTAE 242g Trizma dissolved in 700ml dH2O then add 57.1 ml glacial acetic acid (in corrosive cabinet under hood)next add 100ml 0.5M EDTA (pH 8.0). Adjust final volume to 1 liter.
