Team:BYU Provo/Notebook/Metabolism/mayjune

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==Week of May 10th==
 +
 +
===May 6, 2014===
 +
 +
Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.
 +
 +
===May 8, 2014===
 +
Set up PCR to amplify Bla gene. Used Q5 polymerase and Q5 protocol.
 +
*Q5 PCR
 +
*24.5 ul ddH20
 +
*10 ul Q5 Buffer
 +
*10 ul Q5 Enhancer
 +
*1 ul Bla forward primer BI374
 +
*1 ul Bla reverse primer BI375
 +
*1ul dNTP’s
 +
*1ul Template psB1A3 (resuspended with 10ul ddH20 from iGEM 2013 kit plate 5, well 1G)
 +
*0.5ul Q5
 +
 +
===May 9, 2014===
 +
 +
Ran a gel to check PCR products. Looks great!
 +
Did PCR-up of PCR product using Gen-Elute Kit:
 +
# Add 0.5mL column prep solution to column, spin at 12000 RPM 1 min. Discard eluate after 1 min.
 +
# Transfer PCR reaction mix into the tube
 +
# Add (5x’s volume) of binding solution to 1 volutm of PCR reaction and mix, 250ul
 +
# Add to column, centrifuge at max speed for 1 min
 +
# After 1 min discared eluate, but retain collection tube
 +
# Add 0.5 ml of diluted Wash Solution to the column, centrifuge at max speed for 1 minute
 +
# After 1 minute, discard the eluate, replace the column back in the collection tube. Centrifuge an additional 2 minutes to dry.
 +
# Place the column in a clean tube. Add 50ul elution buffer, wait 1 min. Spin max speed 1 min. Remove column, voila!
 +
 +
Ran restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)
 +
 +
===May 10, 2014===
 +
RD Protocol (repeated for each of the three samples)
 +
*14ul ddH20
 +
*5ul NEB Buffer 4
 +
*0.5 ul 100x BSA
 +
*30 ul DNA sample---(1 each of BBa_J23102, BBa_J23118, and PCR amplified Bla gene)
 +
*1.5 ul each of SpeI HF, and XbaI
 +
 +
Incubated at 37C for 1.5hrs.

Revision as of 21:18, 30 June 2014


BYU 2014 Notebook

Edit May June

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Contents

Week of May 10th

May 6, 2014

Nano-dropped the Promoter plasmids that were recently purified. Almost all of the plasmids were over 100ng/ul concentration.

May 8, 2014

Set up PCR to amplify Bla gene. Used Q5 polymerase and Q5 protocol.

  • Q5 PCR
  • 24.5 ul ddH20
  • 10 ul Q5 Buffer
  • 10 ul Q5 Enhancer
  • 1 ul Bla forward primer BI374
  • 1 ul Bla reverse primer BI375
  • 1ul dNTP’s
  • 1ul Template psB1A3 (resuspended with 10ul ddH20 from iGEM 2013 kit plate 5, well 1G)
  • 0.5ul Q5

May 9, 2014

Ran a gel to check PCR products. Looks great! Did PCR-up of PCR product using Gen-Elute Kit:

  1. Add 0.5mL column prep solution to column, spin at 12000 RPM 1 min. Discard eluate after 1 min.
  2. Transfer PCR reaction mix into the tube
  3. Add (5x’s volume) of binding solution to 1 volutm of PCR reaction and mix, 250ul
  4. Add to column, centrifuge at max speed for 1 min
  5. After 1 min discared eluate, but retain collection tube
  6. Add 0.5 ml of diluted Wash Solution to the column, centrifuge at max speed for 1 minute
  7. After 1 minute, discard the eluate, replace the column back in the collection tube. Centrifuge an additional 2 minutes to dry.
  8. Place the column in a clean tube. Add 50ul elution buffer, wait 1 min. Spin max speed 1 min. Remove column, voila!

Ran restriction digests on vectors and insert. Chose the IGEM plasmids containing a strong, and medium-strong promoter strength respectively)

May 10, 2014

RD Protocol (repeated for each of the three samples)

  • 14ul ddH20
  • 5ul NEB Buffer 4
  • 0.5 ul 100x BSA
  • 30 ul DNA sample---(1 each of BBa_J23102, BBa_J23118, and PCR amplified Bla gene)
  • 1.5 ul each of SpeI HF, and XbaI

Incubated at 37C for 1.5hrs.

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