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Team:Alberta/Notebook/Anchor
From 2010.igem.org
Anchor
Anchor Design
- May 25, 2010:
- Discussed an assembly method: Design a polyA tail Anchor oligo to connect to a polyT tail bound to a cellulose or magnetic bead. The anchor will consist of a primer region, a BsaI cut site, a buffer region, and an A/B prime end. A byte with an appropriate end can be added to this anchor, and another byte can be added onto that, etc. The last thing to be added to the construct is a Terminator byte consisting of a buffer region, BsaI cut site, primer region and a polyT tail. This second primer sequence must be as uncomplimentary as possible to the anchor's primer site to minimize primers binding to each other when PCRing the assembled construct. The primers melting point must be around 65oC because, when the construct is assembled on the bead, it is heated off and the polyA tail of the anchor binds to the polyT tail of the terminator to form a plasmid.
- Used the IDT Analyzer software to determine what length of polyA tail is required for our anchor to have a melting temperature of 30oC (decided to change this melting temp later). This melting temp is sensitive to Na+ and Mg++ concentrations, so referred to ligase buffer and elution buffer for these concentrations.
- Designed an anchor with a polyA10 tail with a primer (Tm=65.1oC) region and the BsaI cut site with an A' end (ACCC).
- May 26, 2010:
- Designed and ordered new anchor oligos. We designed new anchors with larger polyA tail without hairpins, or self dimerization.
- May 27, 2010:
- Ordered new primers for anchor: GCG CGC CCG GTC TCA TGG GTC ACC CTC CC GGG AGG GTG ACC CAT GAG ACC GGC GCG C[A]12
- June 11, 2010:
- Ordered a polyA18 tail because polyA12 tail`s melting temperature is possibly too low.
Anchor Melting Points Test
- June 17, 2010:
- Saturate 40μL beads with 5000 ng of anchor. Wash once with Washing Buffer before adding MilliQ H20 and ligase buffer. Test samples at room temperature (22oC), 30oC, 40oC and 50oC to check anchor’s melting point.
- Results: Nanodrop readings indicate there is DNA present even after wash. Next experiment, wash twice instead of once and measure second wash to confirm whether DNA is present or not. Test both types of anchors and allow annealing of anchors to polydT of beads at a slower rate.
- June 25, 2010:
- Tested polyA12 and polyA18 anchors in ligase buffer and elution buffer following modified mRNA Isolation Protocol. Place in temperatures of 22oC, 25oC, 30oC, 35oC, 40oC, 45oC, 50oC, 55oC, 60oC, 65oC, 70oC, 75oC and 85oC.
- June 28, 2010:
- Nanodrop samples in ligase buffer and elution buffer to plot a graph to determine melting point of anchors.
- June 30, 2010:
- Redo experiment on polyA12 and polyA18 anchors in ligase buffer and elution buffer at higher temperatures: 30oC, 35oC, 40oC, 45oC, 50oC, 55oC, 60oC, 65oC, 70oC, 75oC, 85oC, 90oC and 95oC.
- Nanodrop samples to plot a graph to determine melting point of anchors.
- Results: Compared with the previous graph and used IDT to determined melting point of both anchors. polyA18 Tm=51.3oC and polyA12 Tm=35.2oC
Effects of Bromophenol Blue on Ligation
- July 5, 2010:
- Tested bromophenol blue dilutions of 1/2, 1/8 and 1/32 from a 0.01g/mL stock with digested plasmid to see how it affects ligation. If it does not inhibit ligation or any downstream processing, bromophenol will be useful in visualizing solutions.
- July 6, 2010:
- Results: Bromophenol Blue inhibits ligation.
Anchor Ligating to Byte Test
- July 8, 2010:
- Formulated an equation to calculate the amount of anchor (in moles) to Byte. This formula will be used in all anchor-byte experiments.
- VAnc = VKan (CKan/CAnc) (LAnc/LKan)
- July 10, 2010:
- Tested digestion and ligation of anchor to Byte.
- Results: Anchor does ligate to digested Bytes.
Anchor Binding to Beads Test
- July 13, 2010:
- Calculated volumes required for 20X and 50X anchor excess to bind to cellulose and iron microbeads. Byte was ligated to anchor and the anchor-Byte complex was tested whether it was bound to beads. The ligation of a Byte to anchor is important since the anchor is too small to see on a gel. During the process, we collected flowthrough, washes and elution for cellulose and iron microbeads.
- July 14, 2010:
- Results: High amount of flowthrough, but some binding to iron microbeads only. There was no anchor binding to cellulose beads.
- Re-attempted to bind anchor to cellulose and iron micro-beads, but used only 20X anchor excess and PCR purified to decrease extra anchor not bound to the beads. Also, more beads were used.
- Results: Anchor bound to iron microbeads, but still no binding to cellulose beads.
Bytes Ligating Test with Anchor
- July 20, 2010:
- Calculated volumes of anchor to KanAB and BA Bytes, did a ligation to test the efficiency of Bytes ligating to anchor.
- Results: Lots of smears on the gel.
- July 21, 2010:
- Re-attempt the experiment and apply the second Byte to the Anchor-Byte complex.
- Digested Kan AB and BA Bytes.
- July 23, 2010:
- Gel Extracted digested Kan AB and BA. Bind AB Byte to Anchor by ligation, and bind Anchor with AB Byte to iron microbeads. Apply the second Byte by ligation. There are three types of ligations: ABanch + AB Byte + ligase, ABanch + BA Byte + ligase, and ABanch + BA Byte + no ligase. Flowthrough and elution samples were collected to be run on a gel.
- July 24, 2010:
- Results: Significant excess of anchor present in the AB anch mix. PCR purification is not efficient; therefore, we need to use less than 20X anchor to Bytes in our next assembly experiment.
Assembly Testing
We attempted to create a tetramer consisting of:
- Anchor A, AmpR AB, ori BA, RFP AB and the cap
using the BioBytes method. The parts were joined in the above order and the DNA was recircularized and used to transform cells. The cells were then plated on antibiotic plates.
Result:
Several red colonies formed on the plates, although not enough to be statistically significant
