Difference between revisions of "Team:NYU Shanghai/Protocols"

Revision as of 05:24, 16 September 2015

Project
- Design
- Parts
- Code
- Judging
Team
Outreach
Notebook

Protocols

We built our constructs from digests and ligations of pre-made biobrick parts. Our general conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly.

Recipes

LB-Agar Plates

LB Broth

SOC Media

Making Color

Luciferase

General Overview:

D-Luciferin is too large of a chemical to cross the plasma membrane of e. Coli so cell lysis is required to extract luciferase.
After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.
The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.
Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC

Materials:

D-Luciferin free acid
ATP
MgSO4 · 7H2O
1M HEPES Buffer
Lysozyme
10 mM Tris-HCl

Lysis Buffer:

For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.

Reagent Solution:

Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in polypropylene or glass.

Preparing 1mM D-Luciferin:

Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4 uL of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.

Procedure:
Bacterial lysis:

After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.
Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)
Add 25 uL - 30 uL of lysozyme buffer to the resuspended pellet.
Mix by vortexing for 3 seconds.
Incubate for 2 hours at room temperature.
If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.

Addition of Reagent Solution:

Following the above instructions, prepare a 1mM sample of D-Luciferin.
Following the above recipe, prepare the reagent solution.
In a dark room, add about 250-350 uL of reagent solution to each sample of lysis product.
Light should be emitted within two-three seconds.

Example Calculations:
Lysis Buffer (Desired Total Volume: 15mL)

Chromoproteins

3A Assembly

Transformation

Miniprep

Estimated time

Materials

Biomiga Miniprep Kit

Procedure

Harvest the bacterial culture by centrifugation for 1 min at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely.
Add 250 µL Buffer A1 (Add RNase A to Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting
Add 250 µL Buffer B1, mix gently by inverting the tube 10 times (do not vortex), and incubate at room temperature for 5 minutes.
Add 350 µL Buffer N1, mix completely by inverting/shaking the vial for 5 times and sharp hand shaking for 2 times.
Note: Incubating the lysate in ice for 1 min will improve the yield.
Centrifuge the lysate at 13,000 rpm for 10 minutes at room temperature. If the lysate doesn’t appear clean, reverse the tube angle, centrifuge for 5 more minutes and then transfer the clear lysate to DNA column.
Carefully transfer the clear lysate into a DNA column with a collection tube, avoid the precipitations, spin at 13,000 rpm for 1 minute, discard the flow-through and put the column back to the collection tube.
Add 500 µL Buffer KB into the spin column, centrifuge at 13,000 rpm for 1 minute. Remove the spin column from the tube and discard the flow-through. Put the column back to the collection tube.
Add 650 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 13,000 rpm for 1 minute at room temperature. Remove the spin column from the tube and discard the flowthrough.
Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm.
Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.

Restriction Digest

Ligation

Gel Electrophoresis

Gel Extraction

Estimated time:

Materials
MinElute Midi Gel Extraction Kit

Procedure

Pre-weigh a 2.0mL microcentrifuge tube
Excise DNA fragment from agarose gel with clean, sharp scalpel. Place in the pre-weighed microcentrifuge tube. Minimize the exposure of the DNA samples under UV and amount of gel cut with DNA.
Weigh the tube and calculate the weight of the gel. For every 1 volume of gel (100mg = 100µl) add 3 times the volume of Buffer QG. The maximum amount of gel slice per spin column is 400mg.For >2% agarose gels, add 6 volumes of Buffer QG.
Incubate at 50ºC for 10 minutes (or until the gel has completely dissolved). Vortex the tube every 2-3 minutes during incubation to help dissolve the gel. If color is orange or violet after gel slice has dissolved, add 10µl 3M sodium acetate, pH 5.0. The color of the mixture will turn to yellow.
Add 1 gel volume of isopropanol to the sample and mix by inverting.
Place a MinElute spin column in a provided 2mL collection tube. Apply the sample in the 2.0mL microcentrifuge tube to the MinElute column and centrifuge for 1 min at 13,000 rpm. Discard the flow-through and place the MinElute column back into the same collection tube. If the sample volume exceeds 800µl, simply reload and spin again.
Add 500µl Buffer QG to the MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube.
Add 750µl Buffer PE to MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube. If the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation, let the column stand 2-5 minute after addition of Buffer PE.
Centrifuge the column in the 2mL collection tube for 1 minute at 13,000 rpm again to remove residual ethanol.
Place each MinElute column into a clean 1.5mL microcentrifuge tube. To elute DNA, add 10µl Buffer EB or water to the center of the MinElute membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.
Repeat step 12 but this time load the flow-through back to the membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.
Check Gel extraction product with Nanodrop

PCR

PCR Cleanup

tumblr | strikingly | igem.nyushanghai@gmail.com

@@ Line 56: / Line 56: @@
      color: #6db;
    }
+  #lucText b {
+    margin-top: 1em;
    }
 </style>
@@ Line 328: / Line 330: @@
      <p>Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8.  Store substrate solution at -20ºC in polypropylene or glass.</p>
      <b>Preparing 1mM D-Luciferin:</b>
-     <p>Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4 uL of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.</p><br>
+     <p>Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4 uL of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use.</p>
      <b>Procedure:</b><br>
      Bacterial lysis:
@@ Line 345: / Line 347: @@
        <li>In a dark room, add about 250-350 uL of reagent solution to each sample of lysis product.</li>
        <li>Light should be emitted within two-three seconds.</li>
-     </ol><br>
+     </ol>
      <b>Example Calculations:</b><br>
      Lysis Buffer (Desired Total Volume: 15mL)<br>