Team:Arizona State/Notebook/Tim
06/08/16 - EsaR and LuxR Transformation
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-06-08
Wednesday, 6/8/16
Today, I performed a fast transformation of EsaR and LuxR into BL21. I did EsaR too, just in case LuxR didn't work (as it's been problematic in past transformations). Below are the plasmids I used from the "iGEM 2016 - Final Constructs" box:
●
pLux + LuxR 1 MP (182.3 ng/uL)
●
pEsa + EsaR 5 MP (141.8 ng/uL)
I used the following "reaction mixtures" for this transformation:
A | B | C | D | |
1 | EsaR | LuxR | Water Only | |
2 | Water (uL) | 6 | 7 | 10 |
3 | Plasmid (uL) | 4 | 3 | - |
4 | Cells (uL) | 40 | 40 | 40 |
5 | Total Volume (uL) | 50 | 50 | 50 |
Table 1 - EsaR and LuxR Transformation Reaction Mixtures
The volumes of plasmid used above were to ensure that there were at least 500 ng of DNA in each reaction mixture. The reaction mixtures were plated and left to grow overnight in the 37° incubator.
06/09/16 - EsaR and LuxR Liquid Cultures
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-06-09
Thursday, 6/9/16
Here is a picture showing how the plates turned out from yesterday's transformation:
06-08-16 EsaR + LuxR Fast Transformation Plates.JPG
There were colonies for both EsaR and LuxR, and there were no signs of contamination, as indicated by the clean water only plate. LuxR did not have as many colonies as EsaR; however, LuxR tends to grow few colonies, so that is something I should look into.
After examining the plates, I prepared a spot plate and liquid cultures for EsaR and LuxR. Below are general guidelines on how I prepared LB + Amp, the spot plate, and the liquid cultures: (I will write up detailed protocols separately)
LB+Amp
1.
Secure either a fresh bottle of LB from the 4°C walk-in freezer or a used bottle of LB from your bench
2.
Get a tube of ampicillin from the antibiotics box in the Elsa fridge next to Jimmy's bench
3.
Let the amp thaw
4.
Add an amount of amp in uL that matches the amount of LB in mL, i.e. if there's 100 mL of LB in your container, add 100 uL of amp to the LB
5.
Mix the LB and amp together
Spot Plate
1.
Get an amp plate from the 4°C walk-in freezer and warm it up in the 37°C incubator
2.
Label the plate with the plasmid name(s) (EsaR + LuxR spot plate), the strain of bacteria you transformed into (BL21), the word "Amp", your initials (TC), and the date (06-09-16)
3.
Draw a table/grid on the plate that can accommodate all of your samples, labeling each box with the sender/receiver name and corresponding sample number
Liquid Cultures
1.
Secure 15 mL round bottom culture tubes and label them near the bottom of the tubes. I made 4 cultures for each receiver, so I needed 8 tubes in total
2.
Pick colonies by circling around them with Sharpie and labeling them with their corresponding sample numbers. Try to pick colonies that don't have any satellite colonies
3.
Fill each tube with 4 mL of LB + Amp
4.
Touch a pipette tip to your first colony sample
5.
Dip the tip to its corresponding box on the spot plate
6.
Drop the tip into its corresponding tube
7.
Repeat for the rest of the colonies
The spot plate was left to grow overnight in the 37°C incubator, and the liquid cultures were left to grow overnight in the shaking incubator.
07/14/16 - BraI, CerI, and EsaR Transformation
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-14
Thursday, 7/14
Hey Benchling! It's been a while since I've been on here, but I'm back now in the lab doing wet work. :)
Today, I performed a long transformation for BraI, CerI, and EsaR. I chose the long transformation route because EsaR kept failing at the miniprep stage. My hope is that this more robust transformation protocol will result in higher miniprep yields. These are the samples I used for this transformation:
●
BraI 2 - 589 ng/uL
●
CerI 2 - 106 ng/uL
●
pEsa + EsaR 5 - 141.8 ng/uL
Here is the general protocol I used prepared by Cass:
1.
Heat LB+Amp plates in the 37°C incubator
2.
Add 40 uL of DH5α turbo cells to each 1.5 mL tube with the appropriate plasmid and water
3.
Incubate tubes on ice for 10 minutes
4.
Heat shock tubes at 42°C for 35 seconds
5.
Incubate tubes on ice for 2 minutes
6.
Add 750 uL of SOC media to each tube (SOC media can be found in a rack above the shelf where the sterile water and glass beads are stored)
7.
Shake tubes in a 250 mL beaker at 37°C for 1 hour
8.
Spin down the tubes at maximum speed (16.3 x 1000 g)
9.
Remove 200 uL of supernatant from each tube
10.
Resuspend the cells in each tube
11.
Plate 100 uL of the contents of each tube onto the corresponding plate
12.
Incubate plates overnight at 37°C
Below is a table which shows how the reaction mixtures were prepared:
A | B | C | D | E | |
1 | BraI | CerI | EsaR | Water Only | |
2 | Water (uL) | 9 | 5 | 6 | 10 |
3 | Plasmid (uL) | 1 | 5 | 4 | - |
4 | Cells (uL) | 40 | 40 | 40 | 40 |
5 | Total Volume (uL) | 50 | 50 | 50 | 50 |
Table 1 - BraI, CerI, and EsaR Transformation Reaction Mixtures
As always, the plates were stored in the 37°C shaking incubator to grow overnight.
07/15/16 - BraI, CerI, and EsaR Liquid Cultures
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-15
Friday, 7/15
Here are the plates from yesterday's long transformation:
07-15-16 - BraI, CerI, and EsaR Plates.jpg
All three plates had a TON of colonies, with EsaR having a bit less than BraI and CerI. The water only plate did have two colonies on it, but I think that still prompts little concern for contamination.
After examining these plates, I prepared 4 liquid cultures each for the two senders and one receiver. The cultures were left to grow overnight in the 37°C shaking incubator.
07/18/16 - BraI, CerI, and EsaR Miniprep/Gel Verification
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-18
Monday, 7/18
So, this past weekend, I was slammed at work and didn't have the time to perform the miniprep for my cultures. I was worried they were dead, but Cass advised me to go through with the miniprep anyway, and it worked! Before I talk about the miniprep, here's the spot plate I grew for the two senders/single receiver:
07-18-16 BraI, CerI, and EsaR Spot Plate.JPG
As always, I followed a miniprep protocol adapted from the Sigma-Aldrich "GenElute Plasmid Miniprep Kit User Guide," which is given below:
*As a side note, here's where you can find the reagents and expendables you'll need for the miniprep. The liquid reagent bottles conveniently get bigger in size as you get further in the process, kind of like Russian nesting dolls (you'll see what I mean when you put them next to each other):
●
Resuspension solution - In the 4°C fridge under Daniel's bench. I would recommend getting this only when you are ready to add this to the pellets. Make sure to place this solution back in the fridge immediately after use to prevent degradation of the RNAse
●
Lysis solution, neutralization/binding buffer, column preparation solution, and wash solution - In the white bin on the shelf labeled "Sigma Plasmid Miniprep Supplies" across from Ben's bench
●
Binding columns - In the red box labeled "GelElute Plasmid Mini Spin Column" on the shelf labeled "Sigma Plasmid Miniprep Supplies" across from Ben's bench
●
2.0 mL tubes that fit the binding columns - In the drawer labeled "Microfuge Tubes 2.0 mL" in the rolling cabinet under Ben's bench
Now, the actual protocol...
1.
Place a tube of elution solution or water in the 37°C incubator for use in the last step
2.
Remove the pipette tips from each culture tube
3.
Pellet the cultures by centrifuging them in the IEC Centra CL2 centrifuge on Jimmy's bench with the following settings: 3.8 RPM x 1000 for 5 minutes. The pellets for senders will look red, while those for receivers will look yellow
4.
After centrifugation, discard the supernatant. Be careful not to discard any of the pellet, especially if it's a bit liquidy
5.
Resuspend the pellet with 200 uL of resuspension solution in the culture tube
6.
Transfer the resuspended pellet to a new 1.5 mL tube
7.
Lyse the resuspended cells by adding 200 uL of lysis solution. Mix the contents by gentle inversion (6-8 times) and make sure not to allow the lysis reaction to go longer than 5 minutes. The mixture will become viscous and sticky at this point
8.
Precipitate the cells with 350 uL of neutralization/binding solution and gently invert 4-6 times. You will see a white mass start to form in the tube
9.
Pellet the cell debris by centrifuging at maximum speed (16.3 g x 1000) for 10 minutes
a.
During this long centrifugation, prepare the binding columns by placing them in 2.0 mL microcentrifuge tubes
b.
Add 500 uL of column preparation solution to each column
c.
Centrifuge each tube containing a column at max speed for 1 minute
d.
Discard the flow-through liquid
10.
After centrifugation, you will have a clear liquid (lysate) and a white mass stuck to the side/bottom of the tube. Transfer the lysate to the column prepared during centrifugation, making sure not to come in contact with the white mass. Centrifuge the lysate at max speed for 1 minute and discard the flow-through liquid thereafter
11.
Add 750 uL of wash solution to the column and centrifuge at max speed for 1 minute. Discard the flow-through liquid and centrifuge again at max speed for 2 minutes without adding anything to the column
12.
Transfer the column to a new 2.0 mL microcentrifuge tube and add 50 uL of elution solution or water, which was warmed in the incubator since step 1. Let the column sit for 1 minute and then centrifuge at max speed for 1 minute
13.
Discard the column, but do NOT discard the liquid (eluate), as it now contains the plasmid DNA
14.
Determine the concentrations of your samples using the NanoDrop instrument
Here are the results of this miniprep:
A | B | C | D | |
1 | BraI | CerI | EsaR | |
2 | 1 | 222.083 | 274.153 | 55.914 |
3 | 2 | 240.017 | 69.077 | 78.621 |
4 | 3 | 158.304 | 123.118 | 51.872 |
5 | 4 | 203.633 | 182.236 | 191.059 |
Table1
The concentrations for BraI and CerI were good, and given that I've tried miniprepping EsaR three different times, it seems like I'm not going to get any higher concentrations. I suspect the fourth sample for EsaR has a high concentration because I was running out of water to elute the DNA at that point and probably only used about ~10 uL instead of the normal 50.
Following the miniprep, I ran an agarose gel to verify that the DNA obtained from the miniprep was the plasmid we are looking for. I used 4 uL of 1 kb DNA ladder, and for each sample loaded into the gel, I used 4 uL of loading dye and 2 uL of DNA, except for samples 1, 2, and 3 for EsaR, for which I used 3 uL of loading dye and 3 uL of DNA because of their low concentrations.
07/19/16 - BraI, CerI, and EsaR Sequencing
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-19
Tuesday, 7/19
07/20/16 - ExpR Digest/PCR Purification
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-20
Wednesday, 7/20
7/21/16 - ExpR Ligation/Transformation
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-21
Thursday, 7/21
7/22/16 - ExpR Liquid Cultures
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-22
Friday, 7/22
07/23/16 - ExpR Miniprep/Gel Verification
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-23
Saturday, 7/23
07/24/15 - ExpR Sequencing
Made with Benchling
Project: iGEM 2016
Authors: Timothy Chen
Date: 2016-07-24
Sunday, 7/24
