User:Mara Peterson/Notebook/Mara Peterson/2015/12/04

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Overview and Days 1, 2, and 3 Review

  • Overview

Part 1: Biobrick Parts Assembly http://openwetware.org/wiki/Haynes:BioBrick_Method_short

Part 2: Ligation
Day 1: PCR and confirm the ordered pieces size

• If the sizes are right, proceed to: restriction enzymes, ligation, transform, submit parts to the registry

• If not: troubleshoot

Day 2: Check colonies, liquid cultures, streak plates and colony PCR

Day 3: Plasmid prep, digest, run gel and possibly extract fragments

Day 4: Ligation, transform bacteria

Day 5: Liquid cultures, miniprep, digest, gel to check for assembled construct size

  • Day 1 Review 11/28/15

I was able to create Biobrick Parts for the primers used at the cut sites for HA1, EM7-ZeoR, and HPK-HA2. I did this on Benchling.

Protocol: http://openwetware.org/wiki/Haynes:BioBrick_Method_short

  • Day 2 Review 12/1/15

I performed PCR amplification of the parts I will be using in the ligation, using the primers that I had created and ordered.

Part 1: HA1, FPrimer P040 and RPrimer P137, 100bp, Plasmid: DBN007

Part 2: EM7-ZeoR, FPrimer P136 and RPrimer P139, 442bp, Plasmid: MV8

Part 3: HPK-HA2, FPrimer P138 and RPrimer P041, 2072bp, Plasmid: DBN007

Protocol for Phusion PCR: http://openwetware.org/wiki/Haynes:PCR_clip_clone

Parts 1 and 2 Used the Following PCR Cycle:

98°C, 3 min

35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 30 sec]

72°C, 10 min

4°C ∞

Part 3 (the biggest part) Used the Following PCR Cycle:

98°C, 3 min

35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 60 sec]

72°C, 10 min

4°C ∞

After PCR was complete, I used gel electrophoresis on a 1% agar plate with SYBR to confirm the sizes of the parts. Parts 1 and 2 were successfully amplified and showed up on the gel at around 150bp and 500bp, respectively. Part 3 did not show up on the gel. File:2015-12-01 gel.tif


  • Day 3 Review 12/4/15

I performed PCR Cleanup on Parts 1 and 2, measuring the concentrations before and after to determine the cleanup efficiency.

Part 1: 678 ng/μL before, 6.1ng/μL after

Part 2: 852 ng/μL before, 37.8ng/μL after

I decided to repeat PCR on all 3 parts to improve efficiency and troubleshoot for getting Part 3 to successfully amplify.

PCR for Part 3 will be used with the following protocol, using 0.5μL of DNA and 31.5μL of water for the 50μL total volume. Phusion Protocol: https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530