Difference between revisions of "Team:Arizona State/Experiments"

*Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.

Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.
Set up the following reaction in a microcentrifuge tube on ice:

Component	Volume (uL)
10X T4 DNA Ligase Buffer	2
Vector DNA: 50 ng	X
Insert DNA : 37.5 ng	X
Nuclease Free Water	17
T4 DNA Ligase	1
Total	20

Gently mix the reaction by pipetting up and down and microfuge briefly
For cohesive sticky ends, incubate at 16 C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 1 C overnight or room temperature for 2 hours
Heat inactivate at 65 C for 10 minutes
Chill on ice and transform 1-5 uL of the reaction into 50 uL of competent cells

Vector Digest Protocol for Modular Receiver Vector

Materials

Vector DNA
10x Fast Digest Buffer + Green Dye
Restriction Enzymes
BbsI
SpeI
Deionized Water

Procedure

Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume 50 µl).
Set up the following digest reaction on ice:

Components	Volume (µL)
dH2O	25
10X Fast Digest Buffer + Green Dye	3
MRV Vector	10
BbsI	1
SpeI	1

Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
Incubate

Brianna Lopez

Mini-Prep Protocol

Harvest Cell :

• Pellet 1-5 mL of an overnight recombinant E. coli culture by centrifugation. -The optimal volume of culture to use depends upon the plasmid and culture density.

For best yields for E. coli grown in LB:
use 1-3 mL of culture for high copy plasmids
use 1-5 mL of culture for low copy plasmids
for best yields for E. coli grown in TB or 2X YT:
use only 1 mL of culture

Completely resuspend the bacterial pellet with 200 µL of the Resuspension Solution.
Vortex or pipette up and down to thoroughly resuspend the cells until homogenous

Lyse the resuspended cells by adding 200 µL of the Lysis Solution
Mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous (Don’t vortex)
Do not allow the lysis reaction to exceed 5 minutes

Precipitate the cell debris by adding 350 µL of the Neutralization/Binding Solution
Gently invert the tube 4-6 times
Pellet the cell debris by centrifuging at ≥ 12,000 x g or maximum speed for 10 minutes
Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out as a cloudy, viscous precipitate.
If the supernatant contains a large amount of floating particulates after centrifugation, re-centrifuge the supernatant

Insert a GenElute Miniprep Binding Column into a provided micro-centrifuge tube, if not already assembled
Add 500 µL of the Column Preparation Solution to each miniprep column and centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute
Discard the flow-through liquid

Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute.
Discard the flow-through liquid

Add 750 µL of the diluted Wash Solution to the column
Centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute
Discard the flow-through liquid and centrifuge again at maximum speed for 1-2 minutes without any additional Wash Solution to remove excess ethanol

Transfer the column to a fresh collection tube. Add 100 µL of Elution Solution or molecular biology reagent water to the column
For DNA sequencing and other enzymatic application, use water or 5 Mm Tris-HCL, Ph 8.0, as an eluant
Centrifuge at ≥ 12,000 x g for 1 minute
Use immediately or store at -20˚C

Amber Mani

Colony PCR and Colony Prep

Materials and protocol for running the colony PCR. PCR is used to reproduce (amplify) selected sections of DNA or RNA for analysis.

Materials:

2x Master Mix
Forward and reverse primers
DNA template that will be used
Water
Agar plate(s)

Procedure:

Follow these steps:

Defrost template, 2x Master Mix and primers
Determine desired total working volume (~20-25uL per PCR tube)
Label reaction tubes
Create below reaction mix:
Use this template for the amounts and concentrations to use for making the master mix

Next steps after you have the newly made Master Mix:

Pipette 20 μl of newly mixed green solution into each PCR tube
Colony Prep: to grow more of the sample that you took a colony from for the colony PCR. Can be used to have backup plates when you get a positive result from PCR gel.
Scraped one colony at a time from the existing plate and gently placed the colonly onto the new plate with the labeled spots. Disposed of each tip used into the cooresponding PCR tube labeled to match the placement number on the new plate.
Used empty pipette to attach to each tip in the PCR tubes and pump up and down to mix and remove as much of the leftover bacteria from the tip as possible so it mixed into the PCR tube. Then disposed of the tips in dry waste.
Place the new plates in the incubator and remove the next day to freeze.
Place PCR tubes with the new DNA samples in thermal cycler using the appropriate settings

Growing samples in culture tubes for growth curves

This is used for growing up colony samples overnight for use in the plate reader.

Materials:

15ml culture tubes
LB (AMP or CHLOR)
Grown colonies ready to use on petri dishes
going to use micropipette to grab one colony at a time and add to LB in culture tubes

Procedure:

Collecting colonies and prepping the culture tubes

Best to do this in the evening so the cultures don't grow too long

Label each culture tube with the contents, date and your name
Add LB (AMP or CHLOR) to the culture tube. (general rule for how much LB - use about 1/4 the container volume. If tube is 15ml use about 3-4ml LB)
Take pipette and use a clean tip to collect ONE colony from the plate. Discard the tip INTO the culture tube with the colony on the tip.
Place the lid on the culture tube (not too tight so there is room for gas exchange)
Place the culture tubes into the shaking (37F) incubator for overnight growth.
In the morning you can use the Synergy plate reader to check the OD of the sample and dilute or add fresh LB as needed for the test you are running.

Making and running a gel

What you need and how much of it to make and run an agarose gel

Materials:

* 60 ml of TAE liquid into the flask for gel making.
* 0.6g of the agarose powder to the flask and swirled
* 6.0 μl of the Syber Safe DNA gel stain to the flask and swirled

Procedure:

How to make gel using ingredients

Gel making/running protocol used:

Started with adding 60 ml of TAE liquid into the flask for gel making.
Add 0.6g of the agarose powder to the flask and swirled
Use the microwave to heat the mixture (1 min, swirl and repeat) till all pieces dissolved and was a pure clear liquid
Add 6 μl of the Syber Safe DNA gel stain to the flask and swirled
Carefully poured the mixture into a blank gel tray with spacers in place.
Once the gel is cooled, add the KB+ DNA ladder to the first slot, 2nd slot is left blank and each following slot is to be loaded with 5 μl of each sample.
Once the slots are filled place the gel (still inside the plastic gel holder) into the Galileo/ BioRad electric current machine. Run for approximately 45 minutes at 110V.
To image the gel once finished, use the SynGene gel imager machine.

Chris Connot

Plate Induction & Imaging Protocol

Introduction

This protocol will lead you through the steps of setting up induction plates for Sender/Receiver combos as well as the imaging of the agar plate.

Materials

Agar plates
Sender/receiver bacteria on plates (suggested to be freshly transformed)
Positive/negative controls (GFP+/-Receiver)
MicroPipetter (05 ul to 200ul)
Pipette Tips
GeneSys Machine

Procedure

Induction Plate Setup

Procure agar plates and warm to 37℃.
Procure plates with desired bacteria (senders,receivers,positive/negative control)
design/print out template (fig. 1) for induction test

Fig 1: Induction Plate Layout

Using a micropipettor and a sterile tip, bend the tip on the back of the lid of the agar plate

Fig 2: Bent Tip Method

Use the bent tip to scrape bacteria from any of the required plates (sender/receiver, pos/neg controls)
Paint the bacteria (liberally) on the plate in the designated sections.
Be sure to add enough bacteria so that there is a semi-thick layer that is continuous throughout the designated area.

Imaging the Induction plates

Put magnetic cover on top of UV light ( will have to adjust placement after imaging)
Take off Lid of Culture plate

Fig 3: Prepping Culture Plate for imaging

Fig 4: SynGene Settings

Place culture plate on top of magnetic cover and close the tray. (Fig. 3)
Machine Settings: (Fig. 4)

Blots: Fluorescent Blot
Alexa 488
Epi Mid Wave UV
UV06

Press Green arrow once machine is done calibrating
Press Capture
Save as .sgd in order to have the original data still available to be able to save as .tif
If 3D imaging is needed:

click edit on the lower right side of the screen and click 3D on following screen (upper left)

If annotations are needed

proceed to the edit screen
click on annotate option in upper left hand corner
then place text over parts of the image you want to be annotated
save image in preferred format "as displayed" to save annotations.

Troubleshoot

Christina Smith

Xylaan Livingstone

What should this page contain?

Protocols
Experiments
Documentation of the development of your project

@@ Line 188: / Line 188: @@
 <h2> <ins> Amber Mani </ins> </h2>
+<h2>Colony PCR and Colony Prep</h2>
+<p>Materials and protocol for running the colony PCR. PCR is used to reproduce (amplify) selected sections of DNA or RNA for analysis. </p>
+<h3>Materials:</h3>
+<ul>
+<li> 2x Master Mix </li>
+<li>Forward and reverse primers </li>
+<li> DNA template that will be used</li>
+<li>Water </li>
+<li>Agar plate(s) </li>
+</ul>
+<p>Procedure:</p>
+<h3>Follow these steps: </h3>
+<ol type="1">
+<li> Defrost template, 2x Master Mix and primers </li>
+<li>Determine desired total working volume (~20-25uL per PCR tube)</li>
+<li> Label reaction tubes</li>
+<li>Create below reaction mix:</li>
+<li>Use this template for the amounts and concentrations to use for making the
+master mix</li>
+</ol>
+<h3>Next steps after you have the newly made Master Mix: </h3>
+<ol type="1">
+<li> Pipette 20 μl of newly mixed green solution into each PCR tube </li>
+<li>Colony Prep: to grow more of the sample that you took a colony from for
+the colony PCR. Can be used to have backup plates when you get a
+positive result from PCR gel. </li>
+<li>Scraped one colony at a time from the existing plate and gently placed the
+colonly onto the new plate with the labeled spots. Disposed of each tip used into
+the cooresponding PCR tube labeled to match the placement number on the new
+plate. </li>
+<li>Used empty pipette to attach to each tip in the PCR tubes and pump up and
+down to mix and remove as much of the leftover bacteria from the tip as possible
+so it mixed into the PCR tube. Then disposed of the tips in dry waste.</li>
+<li> Place the new plates in the incubator and remove the next day to freeze.</li>
+<li>Place PCR tubes with the new DNA samples in thermal cycler using the
+appropriate settings </li>
+</ol>

Difference between revisions of "Team:Arizona State/Experiments"

Revision as of 00:11, 29 September 2017

Arizona_State

Protocols

Christina Smith

Ligation Protocol with T4 DNA Ligase

Materials:

Procedure

Vector Digest Protocol for Modular Receiver Vector

Materials

Procedure

Brianna Lopez

Mini-Prep Protocol

Harvest Cell :

Amber Mani

Colony PCR and Colony Prep

Materials:

Follow these steps:

Next steps after you have the newly made Master Mix:

Growing samples in culture tubes for growth curves

This is used for growing up colony samples overnight for use in the plate reader.

Procedure:

Collecting colonies and prepping the culture tubes

**Best to do this in the evening so the cultures don't grow too long**

Making and running a gel

What you need and how much of it to make and run an agarose gel

Materials:

Procedure:

How to make gel using ingredients

Gel making/running protocol used:

Chris Connot

Plate Induction & Imaging Protocol

Introduction

Materials

Procedure

Imaging the Induction plates

Troubleshoot

Christina Smith

Xylaan Livingstone

What should this page contain?

Inspiration

Best to do this in the evening so the cultures don't grow too long