Biomod/2017/StJohns:Results
Results
NOTE: Unless otherwise stated, the agarose gels run (Fig. 12-19) were 0.5X TBE ~10mM Mg2+ 1% denaturing, with 0.5X TBE 10mM Mg2+ buffer, run at 25°C and 450-700 volthours.
All gel images have been uploaded in JPEG form, but TIFF forms can be made available upon request.
Effect of Trehalose on VLP Binding
![Figure 23](https://s3-us-west-2.amazonaws.com/oww-files-public/e/e2/Figure_21_edited_jpeg_Image_CC1_Triangle_MS2_binding_old_vs_new_June_10_2016-DNA_marker_Syber.jpg)
1% Agarose gel; 650 Vhrs
Lanes
- 0.05 pmol Triangle blunt
- 0.05 pmol Triangle blunt + 0.20 pmol T21 VLP
- 0.05 pmol Triangle sticky
- 0.05 pmol Triangle sticky + 0.20 pmol wild type VLP
- 0.05 pmol Triangle sticky + 0.20 pmol old T21 VLP
- 0.05 pmol Triangle sticky + 0.20 pmol new T21 VLP (fresh)
- 0.05 pmol Triangle sticky + 0.20 pmol new T21 VLP (1 week frozen)
Tightening of Bands as a Result of Removing Hinge Staples
![Figure 24](https://s3-us-west-2.amazonaws.com/oww-files-public/4/47/Figure_13-NClaw_and_Capsid%2C_Varying_stoichiometric_ratios_Gel..jpg)
1% Agarose; 700 Vhrs
Lanes:
- 0.05 pmol hinged claw blunt
- 0.05 pmol unhinged claw blunt
- 0.05 pmol hinged claw sticky
- 0.05 pmol unhinged claw sticky
![Figure 24a](https://s3-us-west-2.amazonaws.com/oww-files-thumb/2/2b/Hinged_vs_Unhinged_Claws.png/600px-Hinged_vs_Unhinged_Claws.png)
Distinction Between the Bound and Unbound Version of Unhinged Claw to VLP
![Figure 25](https://s3-us-west-2.amazonaws.com/oww-files-public/7/75/Edited_Image_CC3_NClawX_B_S_bind_sybr_June_22_2017-DNA_Marker_SYBR_Gold.jpg)
1% Agarose; 690 Vhrs
Lanes:
- 0.05 pmol unhinged claw blunt
- 0.05 pmol unhinged claw blunt + 0.20 pmol T21 VLP
- 0.05 pmol unhinged claw sticky
- 0.05 pmol unhinged claw sticky + 0.20 pmol T21 VLP
FRET Signaling of Bound and Unbound Claw
![Figure 26](https://s3-us-west-2.amazonaws.com/oww-files-public/1/19/Edited_Image_CC4_NClawX_D_A_F_bind_June_30_2017-Atto_550_Fret.jpg)
1% Agarose; 680 Vhrs
Lanes:
- 0.05 pmol unhinged claw sticky (donor only)
- 0.05 pmol unhinged claw sticky (donor only) + 0.20 pmol T21 VLP
- 0.05 pmol unhinged claw sticky (acceptor only)
- 0.05 pmol unhinged claw sticky (acceptor only) + 0.20 pmol T21 VLP
- 0.05 pmol unhinged claw sticky (donor+acceptor)
- 0.05 pmol unhinged claw sticky (donor+acceptor) + 0.20 pmol T21 VLP
Yield Produced by Cross-Linking
![Figure 27](https://s3-us-west-2.amazonaws.com/oww-files-public/b/b6/Alison_pics_biomod_1.jpg)
20% Denaturing Acrylamide; 400 Vhrs
Lanes:
- Marker
- 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 0min)
- 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 30 min)
- 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 90 min)
- 0.05 pmol Triangle + 25 mmol 8 MOP (Crosslinked 270 min)
- 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 0 min)
- 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 30 min)
- 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 90 min)
- 0.05 pmol Triangle + 5 mmol 8 MOP (Crosslinked 270 min)
- Plasmid
Increased Reaction Time Led to Increased Crosslinking Yield
![Figure 28](https://s3-us-west-2.amazonaws.com/oww-files-public/2/24/Figure_27.png)
Binding triangle with fluorescently labeled DNA strand 40%
![Figure 29](https://s3-us-west-2.amazonaws.com/oww-files-thumb/1/14/Figure_26_edited.png/600px-Figure_26_edited.png)
1% Agarose; 680 Vhrs
Lanes:
- ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
- ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
- ~0.05pm Uncrosslinked Triangle + T21 Fluorescent Strand
- ~0.05pm Crosslinked Triangle (270 min) + T21 Fluorescent Strand
- ~0.05pm Crosslinked Triangle (270 min) + T21 Fluorescent Strand
- ~0.05pm Crosslinked Triangle (480 min)+ T21 Fluorescent Strand
Purity Check and Quantifying Methylene Blue Strands
![Figure 30](https://s3-us-west-2.amazonaws.com/oww-files-thumb/d/d3/1%29Edited_Image%28MZ%29-Purity_check_and_quantifying_Methylene_Blue_strands.jpg/600px-1%29Edited_Image%28MZ%29-Purity_check_and_quantifying_Methylene_Blue_strands.jpg)
20% Acrylimide Gel; 794 Vhrs (Imaged using scan for MB before staining)
Lanes:
- 10 bp Ladder
- 5.0 pmol (TCA)7 MB
- 5.0 pmol T21 MB
- 0.5 pmol (TCA)7 MB
- 0.5 pmol T21 MB
- 0.05 pmol (TCA)7 MB
- 0.05 pmol T21 MB
- 5.0 pmol A21 C10
- 5.0 pmol (TCA)7 MS2
- 0.5 pmol Claw XII ATTO
- Denaturing Dye
- Blank
Quantifying Methylene Blue strands on Triangle
Prestain
![Figure 31](https://s3-us-west-2.amazonaws.com/oww-files-public/a/ac/2.a%29Edited_Image%28MZ%29-Quantifying_Methylene_Blue_strands_on_Triangle_%28Prestain%29.jpg)
1% Agarose Gel; 487 Vhrs (Imaged using scan for MB before staining)
Lanes:
- 1kb Ladder
- 0.05 pmol Triangle Blunt + 5.0 pmol T21 MB
- 0.05 pmol Triangle Blunt + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(7) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(7) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(7) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
SYBR Stain
![Figure 32](https://s3-us-west-2.amazonaws.com/oww-files-thumb/2/2f/2.b%29Edited_Image%28MZ%29-Quantifying_Methylene_Blue_strands_on_Triangle_%28Stained%29.jpg/600px-2.b%29Edited_Image%28MZ%29-Quantifying_Methylene_Blue_strands_on_Triangle_%28Stained%29.jpg)
1% Agarose Gel; 487 Vhrs (SYBR stain)
Lanes:
- 1kb Ladder
- 0.05 pmol Triangle Blunt + 5.0 pmol T21 MB
- 0.05 pmol Triangle Blunt + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(7) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(7) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Up Triangle(7) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
Attachment of MB strands to Side-Triangle
Prestain
![Figure 33](https://s3-us-west-2.amazonaws.com/oww-files-public/8/8b/3.a%29Edited_Image%28MZ%29-Triangle_binding_to_Methylene_Blue_strands_and_successfully_displaying_a_gel_shift_%28Prestain%29.jpg)
1% Agarose Gel; 581 Vhrs (Imaged using scan for MB before staining)
Lanes:
- 1kb Ladder
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Side Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Side Triangle(15) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
SYBR Stain
![Figure 34](https://s3-us-west-2.amazonaws.com/oww-files-thumb/4/42/3.b%29Edited_Image%28MZ%29-Triangle_binding_to_Methylene_Blue_strands_and_successfully_displaying_a_gel_shift_%28stained%29.jpg/600px-3.b%29Edited_Image%28MZ%29-Triangle_binding_to_Methylene_Blue_strands_and_successfully_displaying_a_gel_shift_%28stained%29.jpg)
1% Agarose Gel; 581 Vhrs (SYBR stain)
Lanes:
- 1kb Ladder
- 0.05 pmol Up Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Up Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Side Triangle(15) A21 + 5.0 pmol T21 MB
- 0.05 pmol Side Triangle(15) A21 + 5.0 pmol (TCA)7 MB
- 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol T21 MB
- 0.05 pmol Side Triangle(15) (TGA)7 + 5.0 pmol (TCA)7 MB
Fluorescence Polarization Results
![Figure 35](https://s3-us-west-2.amazonaws.com/oww-files-thumb/8/85/Carlotta_image_1.png/600px-Carlotta_image_1.png)
![Figure 36](https://s3-us-west-2.amazonaws.com/oww-files-thumb/6/6a/Carlotta_image_2.png/600px-Carlotta_image_2.png)
![Figure 37](https://s3-us-west-2.amazonaws.com/oww-files-thumb/d/d4/Figure_36_vary_vlp_amount.png/600px-Figure_36_vary_vlp_amount.png)