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

• 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

1. Resuspend Cells (verify that appropriate volume RNase A solution was added to the Resuspension Solution

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

2. Lyse Cells

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

3. Neutralize

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

4. Prepare Column

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

5. Load cleared lysate

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

6. Wash Column (verify that ethanol has been added to the bottle of Wash Solution 2)

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

7. Elute DNA

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

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

Insert here

Troubleshoot

Tina

Xylaan

What should this page contain?

Protocols
Experiments
Documentation of the development of your project

@@ Line 104: / Line 104: @@
 <h4> What you need and how much of it to make and run an agarose gel </h4>
 <p>
-Materials:
+<h4> Materials: </h4>
-<ul>
+<ol type="1">
 <li> * 60 ml of TAE liquid into the flask for gel making. </li>
 <li> * 0.6g of the agarose powder to the flask and swirled </li>
 <li>* 6.0 μl of the Syber Safe DNA gel stain to the flask and swirled </li>
+</ol>
 Procedure:
 </p>
 <h3> How to make gel using ingredients </h3>
 <h4> Gel making/running protocol used: </h4>
-<ul>
+<ol type="1">
 <li> Started with adding 60 ml of TAE liquid into the flask for gel making. </li>
 <li> Add 0.6g of the agarose powder to the flask and swirled </li>
@@ Line 126: / Line 127: @@
 V. </li>
 <li> To image the gel once finished, use the SynGene gel imager machine. </li>
-</ul>
+</ol>

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

Revision as of 02:17, 22 September 2017

Arizona_State

Experiments

Tina

B-Lo

Mini-Prep Protocol

Harvest Cell :

Amber

Growing samples in culture tubes for growth curves

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

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:

How to make gel using ingredients

Gel making/running protocol used:

Chris

Troubleshoot

Tina

Xylaan

What should this page contain?

Inspiration

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