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- Acetate precipitation is still used to digest some of our parts out:
- Digestion: J23119 w/ Eco Xba 2
- RBS 2 Xba buffer 2
- RBS 2 Xba buffer 2
- RBS 3 Xba buffer 2
- Finished acetate precipitate of 3 RBS digestions
- Digestion: RBS 1, 2, 3 with Pst in buffer 3
- I15010 & B0015 w/ Xba & Pst in buffer 3
- ‘1’ (I13453) w/ ‘2&3’ is successfully ligated; this means that Part A is done!
- ‘2 & 4’ was ligated to ‘7’ (B0015), but has colonies on the vector controls. Thus, a
PCR screen is performed. We then found 4 colonies that were good and made glycerol stocks of them.
- ‘5’ (J23119) was ligated to ‘2&4&7’, but also had colonies on the vector controls. Thus, a PCR screen is performed. 4 good colonies were cultured and had glycerol stocks.
- Part C is complete as well!
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Meanwhile, we still have to crack down on the issue of not being to cut some of our parts out:
We have spec’d the miniprep, and the miniprep concentrations are relatively high. So we did not start out with low concentrations.
After running a diagnostic gel, however, results showed that digestion failed, we have 2 options:
1) Cut parts in buffer 3 + BSA @ 37C
2) Get high fidelity Pst and cut in buffer 4
Testing enzymes:
Have 3 samples:
1) Cut miniprep: 5 uL template, 1 uL BSA, 1 uL Buffer X, 0.8 uL Enzyme, 1.2 uL H20
2) Same as 1, but with other enzyme
3) 10 uL template, 2 uL buffer 3, 2 uL BSA, 1.2 uL Xba, 0.8 uL Pst, 1 uL H2O
C0051: Xba in buffer 4, Pst in buffer 3, Xba + Pst
E1010: Xba in buffer 4, Pst in buffer 3, Xba + Pst
Result of enzyme test is that…
Pst is not cutting well!! On diagnostic gel, there was smearing every time Pst was used to cut. Thus, an order for new Pst has been placed.
There are still some issues with the C0051 part of the initiator, however:
In the words of Jennifer Lau, “I have no explanation for what’s going on. Enzymes are cutting, but now I run into a new problem. I’m getting three bands in the upper region of the gel. Not even my entire plasmid is that big.
1) Bad gel? Nope, my gel was smooth and had no bubbles in it
2) C0051 had bands larger than the entire plasmid length itself!!”
Hypothesis: the new C0051 colonies that I streaked are contaminated in some way, shape, or form
Will see how new minipreps work out today (from original colonies)
Will do multi-digestions of C0051 (6 of them)
All were cut with same enzymes, but only 1 had bands in the correct places
Cultured this C0051 (called C0051 #2 because only the one in well #2 had successful bands)
Afterward using the new Pst, digestions have been going quite smoothly: RBS-E1010, C0051, F1610, I12006. In the words of Keegan Owsley, “Just extracted these bad boys. Seriously, best bands ever. Except I12006. You were all smeary. But whatever. I forgive you.”
This led to the successful ligation of RBS-E1010-F1610 and RBS-C0051 (finally!) (RBS-E1010 had been successfully ligated earlier in the week.)
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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