Team:Wageningen UR/Journal/week18

From 2012.igem.org

Revision as of 15:05, 17 September 2012

• Home
• Team
  • The Team
  • Team Registration
• Project
  • 1. Introduction
  • 2. PnA System
  • 3. Virus-Like Particles
    • CCMV
    • HepB
    • Polerovirus Natural BioBrick
  • 4. Outside Modification
  • 5. Inside Modification
  • 6. Detection of VLPs
  • 7. Applications
  • 8. The Constructor
  • 9. Final Overview
• Human Practices
  • Human Practices Home
  • 1. Mini Symposium
  • 2. Visiting Secondary Schools
  • 3. Discovery Festival
    • Communication Science
  • 4. Stakeholders
  • 5. Munich CAS Conference
• Safety
  • Introduction
  • 1. General safety
  • 2. Virus-related safety
  • 3. Regulations
  • 4. Safety suggestions
  • 5. Safety of applications
• Modeling
  • Human Body Model
  • Tertiary folding prediction
  • VLP assembly model
• Attributions
• Parts
• Notebook
  • Journal
  • Protocols

---

Last year's project: Synchronized Oscillatory System

iGEM Home

Contact Us

Our Sponsors:

Hepatitis B

27.August

• Miniprep of HepB+IPTG in BBa_J04450 backbone in DH5α (transformation from 20 August) to check again wheather there is an insert

-> the miniprep had a very low yield

28 August

• EM detection of VLPs

After explanation about the electron microscope, we examined different samples amongst which the HepB sample prepared on August 16. The EM confirmed that indeed VLPs were formed.

• Miniprep of HepB PCR product in BBa_J04450 backbone from colony PCR 20.Aug and HepB with his-tag (in BBa_J04450 backbone) from colony PCR 20Aug to check again if there is an insert (since colony PCR is not working well sometimes)

Hepatitis B outside modification

• 27 August (Kees)

• Whole plasmid PCR

Instead of using the general FW and Rev primers, the whole plasmid can be amplified alternatively. This allows a more controlled Tm and only 1 amplicon instead of 2 which have to be combined. The same steps 1-4 are still required.

First try with Pfusion polymerase did not have any result.

• 29 August (Kees)

• Gradient PCR

A greadient pcr was performed using Taq polymerase. This can only determine wether the primers do work at a certain, yet for phusion undertermined temperature. Besides, Taq adds an A, causing a mutation in the final step. Therefore, the products can not be used, even if we accept the absence of proofreading in Taq.

The bands on the gel are as expected somewhat over 3k bp. This proofs this pcr step is possible.

• Phusion Polymerase PCR

After the proof was given that this first pcr step is possible, we use phusion to obtain usable product. The new annealing temperature is 63˚C, based upon the Fermentas calculation. The elongation time was set to 2min, which should allow elongation of the product at an elongation speed of 1k bp/min. Again, the pcr did not work. Therefore, new primers were ordered which can execute the whole pcr at once, including a TEV-protease sight.

Hepatitis B inside modification

27.August

• Gel extraction of PCR step 3 sample from 24.Aug

• 4th (final) step of the PCR reaction with the sample from the gel purification

-> this step seemed to work - the gel shows a band at the expected size slighly larger than the band from the 3rd step.

• Gel purification of the fragment of step 4 (H4)

28.August

• ligation of the final PCR product (HepB-insidecoil) in Bba_J04500 and transformation with Mach1

GFP modification

27.August

• 2nd try of the check and amplification of GFP-coil PCR product from 14.Aug

-> still no band visible for the GFP check/amplification

• Miniprep of GFP-coil in BBa_J04450 backbone in JM109 (transformation from 20 August) to check again wheather there is an insert