XIE/Enzyme:2025/Dec: Difference between revisions
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== 12.08 == | == 12.08 == | ||
* <big>'''HR-DNA Origami（NO） Folding'''</big> | * <big>'''HR-DNA Origami（NO） Folding'''</big> | ||
*Materials | |||
{| class="wikitable" | {| class="wikitable" | ||
! Material | ! Material | ||
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|} | |} | ||
# | # | ||
# | *Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h. | ||
*Purification (12.09): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add UP Water to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with UP Water, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate. | |||
== 12.09 == | |||
* <big>'''HR-DNA Origami（NO） Folding'''</big> | |||
*Materials | |||
{| class="wikitable" | |||
! Material | |||
! Volume（100 μL） | |||
|- | |||
| Staples (500 nM) | |||
| 25 μL | |||
|- | |||
| P8064 (150 nM) | |||
| 10 μL | |||
|- | |||
| 10X PBS | |||
| 10 μL | |||
|- | |||
| H2O | |||
| 55 μL | |||
|} | |||
# | |||
*Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h. | |||
*Purification (12.10): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add 1X PBS to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with 1X PBS, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate. | |||
== 12.10 == | |||
* <big>'''Agarose Gel Electrophoresis '''</big> | |||
*'''Gel Preparation''' | |||
#Recipe: 2.4 g agarose in 120 mL 0.5× TBE | |||
#Heat in a microwave to dissolve, cool slightly, adjust volume with 0.5× TBE to 120 ul, then add 1.2 mL of 1 M MgCl2 and 12 µL GoldView DNA stain. | |||
#Insert comb and waite for the gel to cool and solidify. | |||
*'''Sample preparation''' | |||
#Origami (15 nM): sample:loading buffer = 5:1. | |||
#Scaffold: take 1 µL scaffold, dilute to 10 µL (final concentration 15 nM); scaffold:loading buffer = 5:1. | |||
*'''Electrophoresis''' | |||
#Run at 90 V for 120 min in 0.5× TBE with 10 mM MgCl2 | |||
#'''Marker、Scaffold（1X）、Origami（10.08-未洗）、Origami（10.08—清洗）、Origami（10.09-未洗）、Origami（10.09-清洗）、Scaffold（10X）、Marker''' | |||
[[FIle:Origami(NO)_25_12_10.png|thumb|center|600px|Origami(NO)]] | |||
Revision as of 10:02, 10 December 2025
12.08
- HR-DNA Origami（NO） Folding
- Materials
| Material | Volume（100 μL） |
|---|---|
| Staples (500 nM) | 25 μL |
| P8064 (150 nM) | 10 μL |
| 10X PBS | 10 μL |
| H2O | 55 μL |
- Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.
- Purification (12.09): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add UP Water to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with UP Water, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.
12.09
- HR-DNA Origami（NO） Folding
- Materials
| Material | Volume（100 μL） |
|---|---|
| Staples (500 nM) | 25 μL |
| P8064 (150 nM) | 10 μL |
| 10X PBS | 10 μL |
| H2O | 55 μL |
- Program folding in PCR thermocycler : The mixed sample was put on a thermal ramp starting with a rapid heat denaturation at 80 °C for 5 min followed by cooling from 80 °C to 60 °C over 20 min, then slow cooling from 60 °C to 24 °C over 14 h.
- Purification (12.10): Reserve a 10 µL aliquot. Add the remaining 90 µL to a 100‑kDa MWCO centrifugal filter unit and dilute to 500 µL with 1× PBS. Centrifuge at 8,000 g for 1.5 min and discard the filtrate . Add 1X PBS to 500 ul to the filter unit and repeat the centrifugation; perform this wash step three times. After the final wash, adjust the volume in the filter unit to 100 µL with 1X PBS, invert the filter unit into a fresh collection tube, and centrifuge at 8,000 g for 1.5 min to collect the concentrate.
12.10
- Agarose Gel Electrophoresis
- Gel Preparation
- Recipe: 2.4 g agarose in 120 mL 0.5× TBE
- Heat in a microwave to dissolve, cool slightly, adjust volume with 0.5× TBE to 120 ul, then add 1.2 mL of 1 M MgCl2 and 12 µL GoldView DNA stain.
- Insert comb and waite for the gel to cool and solidify.
- Sample preparation
- Origami (15 nM): sample:loading buffer = 5:1.
- Scaffold: take 1 µL scaffold, dilute to 10 µL (final concentration 15 nM); scaffold:loading buffer = 5:1.
- Electrophoresis
- Run at 90 V for 120 min in 0.5× TBE with 10 mM MgCl2
- Marker、Scaffold（1X）、Origami（10.08-未洗）、Origami（10.08—清洗）、Origami（10.09-未洗）、Origami（10.09-清洗）、Scaffold（10X）、Marker
_25_12_10.png)