IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-16: Difference between revisions
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(folding experiment) |
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| align="center" style="background:#f0f0f0;"|'''total volume''' | | align="center" style="background:#f0f0f0;"|'''total volume''' | ||
|- | |- | ||
| B|| | | B||9 {{ul}} p7308||16 {{ul}} 3.2.B.core (250 nm)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}} | ||
|- | |- | ||
| C||4.5 {{ul}} p7308|| | | C||9 {{ul}} p7308||16 {{ul}} 3.2.C.core (250 nm)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}} | ||
|- | |||
| 6hb||4.5 {{ul}} p7308||2 {{ul}} 6hb.v5 (0.99 {{um}})||2 {{ul}} 10x||11.5 {{ul}}||20 {{ul}} | |||
|} | |} | ||
Annealing protocol | Annealing protocol | ||
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* 60 cycles: wait 2 minutes, decrease 1{{c}} | * 60 cycles: wait 2 minutes, decrease 1{{c}} | ||
* hold at 4{{c}} | * hold at 4{{c}} | ||
====Latch-closing protocol==== | |||
* pipet 20 {{ul}} of expts B and C into respective clean 0.2 mL PCR tubes | |||
* add 1.0 {{ul}} of 3.2.B and 3.2.C (1.25 mM), respectively | |||
* incubate at room temp. for 30 min. | |||
====Gel analysis==== | |||
* 2% agarose gel supplemented to 10 mM {{mgcl2}} | |||
** 100 mL solution with 5 {{ul}} 10 mg/mL EtBr | |||
* run in 1x TBE supplemented to 10 mM {{mgcl2}} | |||
* 35 min at 130 V | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Lane''' | |||
| align="center" style="background:#f0f0f0;"|'''Contents''' | |||
| align="center" style="background:#f0f0f0;"|'''Loading Buffer''' (10x TBE/glycerol) | |||
|- | |||
| 1||1kb DNA ladder (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
| 2||control: +scaffold -oligos (10 {{ul}} 23 {{um}})||1.1 {{ul}} | |||
|- | |||
| 3||control: -scaffold +3.2.B.core oligos (10 {{ul}} 25 {{um}})||1.1 {{ul}} | |||
|- | |||
| 4||expt B -latches (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
| 5||expt B +latches (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
| 6||control: -scaffold +3.2.C.core oligos (10 {{ul}} 25 {{um}})||1.1 {{ul}} | |||
|- | |||
| 7||expt C -latches (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
| 8||expt C +latches (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
| 9||control: -scaffold +6hb.v5 oligos (10 {{ul}} 25 {{um}})||1.1 {{ul}} | |||
|- | |||
|10||expt 6hb (10 {{ul}})||1.1 {{ul}} | |||
|- | |||
|11||control: +scaffold -oligos (10 {{ul}} 23 {{um}})||1.1 {{ul}} | |||
|} | |||
[[Image:20060716_3.2.B%2CC.jpg|thumb|2% agarose gel electrophoresis]] | |||
'''Results/discussion''' | |||
* expts B and C, with and without latches (lanes 4, 5, 7, 8, respectively), may have folded successfully, and run with approximately the same mobility as [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-7-14#Gel_analysis|expt A]] | |||
* oligos appear very faintly in lanes 3-10 (appeared brighter before photograph was taken) | |||
* expts B and C without latches (lanes 4 and 7) appear to have similar motility to the same expts with latches (lanes 5 and 8, respectively), which is not conclusive as to whether the latch design was successful | |||
* 6-helix bundle (lane 10), as previously observed, has faster motility than p7308 scaffold (lanes 2 and 11) | |||
Latest revision as of 11:31, 16 July 2006
Container v3.2
c3.2 Folding Experiment
Reagents (each expt in respective 0.2 mL PCR tube)
| Experiment | Scaffold | Oligos | Folding buffer | dH2O | total volume |
| B | 9 μL p7308 | 16 μL 3.2.B.core (250 nm) | 4 μL 10x | 11 μL | 40 μL |
| C | 9 μL p7308 | 16 μL 3.2.C.core (250 nm) | 4 μL 10x | 11 μL | 40 μL |
| 6hb | 4.5 μL p7308 | 2 μL 6hb.v5 (0.99 μM) | 2 μL 10x | 11.5 μL | 20 μL |
Annealing protocol
- start at 80[[:Category:{{{1}}}|{{{1}}}]]
- 60 cycles: wait 2 minutes, decrease 1[[:Category:{{{1}}}|{{{1}}}]]
- hold at 4[[:Category:{{{1}}}|{{{1}}}]]
Latch-closing protocol
- pipet 20 μL of expts B and C into respective clean 0.2 mL PCR tubes
- add 1.0 μL of 3.2.B and 3.2.C (1.25 mM), respectively
- incubate at room temp. for 30 min.
Gel analysis
- 2% agarose gel supplemented to 10 mM MgCl2
- 100 mL solution with 5 μL 10 mg/mL EtBr
- run in 1x TBE supplemented to 10 mM MgCl2
- 35 min at 130 V
| Lane | Contents | Loading Buffer (10x TBE/glycerol) |
| 1 | 1kb DNA ladder (10 μL) | 1.1 μL |
| 2 | control: +scaffold -oligos (10 μL 23 μM) | 1.1 μL |
| 3 | control: -scaffold +3.2.B.core oligos (10 μL 25 μM) | 1.1 μL |
| 4 | expt B -latches (10 μL) | 1.1 μL |
| 5 | expt B +latches (10 μL) | 1.1 μL |
| 6 | control: -scaffold +3.2.C.core oligos (10 μL 25 μM) | 1.1 μL |
| 7 | expt C -latches (10 μL) | 1.1 μL |
| 8 | expt C +latches (10 μL) | 1.1 μL |
| 9 | control: -scaffold +6hb.v5 oligos (10 μL 25 μM) | 1.1 μL |
| 10 | expt 6hb (10 μL) | 1.1 μL |
| 11 | control: +scaffold -oligos (10 μL 23 μM) | 1.1 μL |
Results/discussion
- expts B and C, with and without latches (lanes 4, 5, 7, 8, respectively), may have folded successfully, and run with approximately the same mobility as expt A
- oligos appear very faintly in lanes 3-10 (appeared brighter before photograph was taken)
- expts B and C without latches (lanes 4 and 7) appear to have similar motility to the same expts with latches (lanes 5 and 8, respectively), which is not conclusive as to whether the latch design was successful
- 6-helix bundle (lane 10), as previously observed, has faster motility than p7308 scaffold (lanes 2 and 11)
