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 |
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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:
- 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.