We discovered our DNA oligonucleotides had degraded before assembly could be completed.
We spent about one month over the Summer trying to assemble our gBlocks for PCR. During this time we modified the PCR heat cycle many times, believing optimization of the heat cycles to be the issue holding us back. We even reached out to a grad student, Jessica Stieglitz, for tips on PCR optimization. Only after weeks of failed attempts (X PCR’s over X Gels), we approached Jessica Stieglitz again for help, explaining our problem and protocol more specifically, and realized that assembly PCR required one run of the thermocycle without polymerase. By the time we ran the corrected assembly PCR protocol, our DNA blocks were degraded already from so many freeze-thaw cycles and periods outside of refrigeration. Regardless, we did see some bands of properly assembled product, although it was too impure for any use. After that month was over, Aiden Lewis, the treasurer head had to return home, since his stipend for summer housing only lasted one month. While he was gone, an order was placed to order more gBlocks for another trial of the project. However, earlier in the year, the wrong mailing address had been placed and this had not been corrected. Since Aiden was not around to pick up the shipment from the wrong address, and he was on vacation out of the country, the rest of the team failed to receive replacement gBlocks, as they were lost in the mail.
As time was running out, we shifted our focus to completion of the interlab study to fulfill the bronze medal requirement
The interlab study was done successfully and data was submitted in time through the online form. Some data was not consistent with expected results, likely due to confusion over handwritten labels on some cultures.
What went wrong?
- Lack of experience: most team members were Freshmen, or had only participated in one year previously. Tufts iGEM was unable to compete last year due to lab renovations.
- Reluctance to reach out to grad students: we should have asked grad students for a second opinion before assuming we knew what the issue in our protocol was.
- Lack of funding: we should be more proactive next year to try to secure more funding so we can order backup-reagents more quickly, and have longer lasting summer stipends.
- Not enough team coherence: we should have been more communicative with each other to prevent the issue with missed gBlock shipments.
This shows that the Gene Blocks have been degraded, evidenced by smudges: