SBB11Ntbk-JessicaWen

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Jessica Wen 15:07, 27 April 2011 (EDT)

Today we came into lab and made our powerpoint presentation for our project

Jessica Wen 15:07, 21 April 2011 (EDT)

today we came into lab and analyzed our data from the Tecan

Jessica Wen 13 April 2011 (PST)

Rishi obtained the data from the Tecan to format and present later. See Rishi Rawat's notebook for .csv data files. Curve analysis will be performed later.

Jessica Wen 15:20, 12 April 2011 (EDT)

Induce:
--make 250uL 20% arabinose solution:
add 50mg Arabinose to ~.25g LB+Spec
--make 2 mL .2% Arabinose solution:
take 20uL of 20% arabinose solution, add to 1880 uL Lb+Spec
--make 2 mL .02% Arabinose solution:
take 200uL of .2% arabinose solution, add to 1800 uL Lb+Spec
6.9) make stock solutions of arabinose. Using LB+SPEC
%Arabin uL(20% or .2% or .02%) to add to 10mL LB+Spec
0.0 0.0
1.931e-06 .9655
5.517e-06 2.7585
1.576e-05 7.88
4.504e-05 2.252
0.0001287 6.435
0.0003677 18.385
0.00105 52.5
0.003 1.500
0.00858 4.290
0.0245 12.250
0.07 35.0
0.2 100.0


7.) Add Arabinose to a final concentration of: tube# 1t 0.0 2t 1.931e-06 3t 5.517e-06 4t 1.576e-05 5t 4.504e-05 6t 0.0001287 7t 0.0003677 8t 0.00105 9t 0.003 10t 0.00858 11t 0.0245 12t 0.07 13t 0.2

http://www.pnas.org/content/99/11/7373.full- Arabinose Concentrations


8.) Post induction, Load 100uL from each test tube onto a plate to go into a Tecan plate reader- Do this twice per test tube so that all samples are duplicates.

a.) Setup Tecan to measure OD(600nm) of the each sample in the plate every 15-30 minutes overnight

Jessica Wen 17:34, 11 April 2011 (EDT)

Pick colonies from plates in 4 C fridge one of ToxR, Violacein, and control in 3mL LB+Spec media

Jessica Wen 08 April 2011 (PST)

Rishi, Xin Xin, and I came in early but no one in the Anderson lab was there before 10 AM. Experiment scrapped; to be started over later.


Jessica Wen 06 & 07 April 2011 (PST)

Observations 12pm: Rishi Rawat moved cells from incubator to 4C fridge in Anderson lab, next to incubator - some samples suspected to be mislabeled.

All plates had 100s of colonies--> the transformation worked!

However, all of the colonies on the "Violacein" plate were Darkred/purple in color - makes sense, Violacein-expressing bacteria are purple in color

At ~5PM on April 07th, I started overnight cultures for the 3 samples. They are in the 37C shaker in the Anderson Lab

""Overall Protocol""

List of Materials: Bac Culture of: pBCA1256-BCA1144 1766-BCA1144 pBCA9523-BCA1144 ToxR- pBca1256-bdc004-> Plasmid for ToxR named bdc022 Violacein- pBca9523-jtk2914 +Spec LB (200mL) Arabinose x grams MC1061 4 Spec Plates

Jessica Wen 13:34, 5 April 2011 (EDT)

Being Project: Evaluate toxicity of ToxR and Violacein
Team Members:
LIN, XIN XIN
RAWAT, RISHI RAGHAV
WEN, JESSICA TSAI
KESAVARAJU, ANAND

1.) Transform (heatshock) nontoxic gene plasmids into MC1061, plate +Spec
2.) Transform(heatshock) toxic gene plasmids into MC1061,plate +Spec

Procedure

 1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

Jessica Wen 13:23, 10 March 2011 (EST)

the gel from the previous lab did not work. repeat procedure from 3/8/11

  • parts do not show up again, GSI's will redo it over the weekend (thank you!)

Jessica Wen 13:41, 8 March 2011 (EST)

Mapping

Procedure
1. Set up the following 10uL reaction in a PCR tube:

4uL ddH2O
4uL Miniprepped plasmid
1uL 10x NEB Buffer 2
.5uL EcoRI
.5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)

(FOR Righty Methylated Parts use Eco/Xho!)
2. Incubate at 37 on the thermocycler for 30 minutes
3. Run an analytical gel
4. Take a picture of the gel
5. Calculate the expected fragment sizes
Are the calculated sizes consistent with the bands on the gel?

Image:030811Gel.jpg


  • parts do not seem to show up repeat next time

Jessica Wen 13:37, 3 March 2011 (EST)

Four colonies of each part were picked and allowed to culture. All four colonies of each part (sb1112, sb1137, sb1123) cultured successfully except one of the sb1137 colonies.

Miniprep Purification of DNA

Procedure
(using the QIAGEN QIAPrep Spin Miniprep kit)

1. Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube.
2. Dump supernatant
3. Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10. Wash each column with 500 uL of PB buffer.
11. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
12. Wash with 750uL of PE buffer (washes the salts off the resins).
13. Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
14. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
15. Label new Microcentrifuge tubes and put columns in them.
16. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
17. Spin in centrifuge at top speed for 30 seconds.
18. Take out columns and cap the tubes.
19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Jessica Wen 13:37, 1 March 2011 (EST)

Ligation

Procedure
1.Set up the following reaction:

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase

2. Pound upside down on the bench to mix
3. Give it a quick spin to send it back to the bottom of the tube
4. incubate on the benchtop for 30min
5. Put on ice and proceed to the transformation



Transformation

Procedure
1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

Jessica Wen 13:53, 24 February 2011 (EST)

Zymo Cleanup of Digest

Procedure
1. transfer into the Zymo column inside a collection tube (small clear guys)
2. spin through, discard waste.
3. Add 200 uL of A4 Wash Bbuffer (which is basically 70% ethanol)
4. spin through, discard waste.
5. Add 200 uL of A4 Wash Buffer
6. spin through, discard waste.
7. spin for 90 seconds, full speed to dry.
8. elute with water into a fresh Eppendorf tube

Jessica Wen 13:16, 22 February 2011 (EST)

Eco/Bam Digest of PCR Products

Procedure
1. For each part place in a new PCR tube:

  • 8uL PCR Product
  • 1uL NEB Buffer
  • 0.5 uL EcoRI
  • 0.5 uL BamHI

2. Place in thermocycler for 1 hour
3. Run preparative gel

  • cut out bands place in epindorph tube
  • add 600uL ADB buffer and place on heat block at 55 degrees C for 10 min to dissolve

Gel Image (lanes 2-4, sbb1112, sbb1137, sbb1123 respectively) Image:022211-PrepGel4.jpg

Jessica Wen 13:16, 21 February 2011 (EST)

  • Email from Professor Anderson:

"I noticed on Friday that many of you were using the "AW Wash Buffer" for your Zymo cleanups rather than the "A4 Wash Buffer". Unfortunately, they are both called "Wash Buffer" but they are not the same. AW is equivalent to the Qiagen PB Buffer--it has quanidinium chloride in it. What you wanted was A4, which is ethanol/water. So, your DNA is probably now in the landfill.

I redid all your PCRs and Zymo cleanups and threw out all the old tubes. So, you're back on schedule. I've also modified the protocols and have ordered Qiagen minipreps to eliminate the confusion around the two kits. If you are printing out the protocols, please reprint them before doing things. These are the protocols you will be using:

  • Gel Image (sbb1112 - lane 8, sbb1137 - lane 16, sbb1123 - lane 4)

Image:022111-AnalGel2.jpg

Jessica Wen 13:31, 17 February 2011 (EST)

Analytical Gel of PCR Products

Procedure

Gel Preparation In a 0.5mL tube add:
2uL of PCR product
5uL of DNA Loading Buffer

Run Gel

Sb1112 did not work. Run PCR cloning again, one the same as before and one with 10% DMSO.

for sbb1137 and sbb1123 continue with Zymo Cleanup, which removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction.

Zymo Cleanup
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of PE or Zymo Wash buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of PE or Zymo Wash buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Run analytical Gel

  • Gel Image (lanes 2-4, sbb1112, sbb1137, and sbb1123 respectively)

Image:021711-AnalGel2.jpg

Jessica Wen 14:05, 15 February 2011 (EST)

Cloning by PCR of the following construction files

Part sbb1112 {P_cspI}
PCR jwsbb1112F/ss37f on MG1655 (506bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sb1112 {P_cspI}



jwsbb1112F Forward Construction of P_cspI promoter part ccaaaGAATTCatgAGATCTATGAATCTTTAAGAGATAAAAAGC
ss37f BglBrick basic part cloning of cspI promoter tttggGGATCCgccatctttcggcgtgatgaaacc


Part sbb1137 {P_yciG}
PCR ss64r/ss64f on MG1655 gen. (616bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1137 {P_yciG}


ss64fReverse Cloning of P_yciG tttggGGATCCgtcggatgccttctcacggtcttcgg
ss64rForward Cloning of P_yciG aaaccGAATTCatgAGATCTagcagcgattgatgcaggagctgcg


Part sbb1123 {P_rfaH}
PCR ss49r/ss49f on MG1655 gen. (421bp, EcoRI/BamHI)
Sub into pBjh1601KC-Bca1144#5 (EcoRI/BamHI, 3131+910, L)
Product is pBjh1601KC-sbb1123 {P_rfaH}



ss49fReverse Cloning of P_rfaH tttggGGATCCgcatcactgactgcagtacgttttccacgcacg
ss49rForward Cloning of P_rfaH aaaccGAATTCatgAGATCTgcgtgatacgttttagctcaccctg


Procedure
If the oligo is not already in 10uM concentration make an oligo dilution of:
9uL water
100uL uM oligo

Make one PCR reaction tube of the following for each part:
24uL ddH2O
3.3uL 10x Expand Buffer "2"
3.3uL dNTPs (2mM in each)
1uL Oligo 1, 10uM
1uL Oligo 2, 10uM
0.5uL Expand polymerase "1"
0.5uL Template DNA

Jessica Wen 15:08, 10 February 2011 (EST)

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