User:Vlau/Lab Notebook: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
DavidRamos (talk | contribs) No edit summary |
No edit summary |
||
Line 1: | Line 1: | ||
==[[Week 1]]== | |||
==='''06.12.06: Mon.'''=== | |||
====''Folding DNA Nanostructure''s==== | |||
1. Working Stocks | |||
44 nM scaffold (20 microL) | |||
0.99 microM of each oligo | |||
2. Protocol | |||
- goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 microL | |||
- calculations: | |||
scaffold = (10 nM)/(44 nM) * 20 microL = 4.5 microL | |||
oligos = (100 nM)/(990 nM) * 20 microL = 2 microL | |||
- reaction mixture: | |||
4.5 microL p7308 scaffold | |||
2 microL oligos | |||
2 microL 10x folding buffer (500 mM HEPES ph 7.5, 500 mM NaCl, 100 mM MgCl2) | |||
11.5 microL dH2O | |||
- annealing times: | |||
90 dC, 5' | |||
65 dC, 20' | |||
55 dC, 20' | |||
45 dC, 20' | |||
37 dC, 30' | |||
- neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water | |||
====''Transformation''==== | |||
1. Plasmids | |||
R0010: lac operon promoter | |||
E7104: T7 promoter + GFP | |||
E0241: GFP | |||
2. Protocol | |||
- let chemically competent OneShot Top10 cells thaw on ice | |||
- added appropriate DNA to cells and tapped gently to mix | |||
- let sit on ice for 20 min | |||
- heat shocked @ 42 dC for 30" | |||
- let cool on ice for 2 min | |||
- added 200 microL SOC media | |||
- shook @ 37 dC for 1 hr | |||
- pipetted and spread onto agar plates treated w/ ? | |||
- incubated @ 37 dC overnight agar side-up | |||
==='''06.13.06: Tues.'''=== | |||
====''Gel Electrophoresis w/ 2% Gel''==== | |||
1. Protocol | |||
- mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask | |||
- heated for 1.5 min in microwave w/ top loosely screwed on | |||
- added 3 microL EtBr | |||
- poured into gel frame and let set for 20 min | |||
- placed into running box and submerged w/ TBE | |||
- wells loaded | |||
- ran gel @ 130 V, 45 min | |||
2. Samples | |||
L. 1: 1 kB ladder | |||
L. 2 and 3: DNA nanostructure | |||
L. 4 and 5: neg control w/ no oligonucleotides | |||
L. 6 and 7: neg control w/ no scaffold | |||
L. 8: 1 kB ladder | |||
loading dye: BTB dye 50% glycerol + 10X TBE | |||
ladder: 10 microL + 1 microL dye | |||
samples: 10 microL + 1 microL dye | |||
3. Image | |||
[[Image:Pic 1.jpg|thumb|center]] | |||
====''Transformation P. 2''==== | |||
1. Protocol | |||
- prepared 2 liquid cultures for each transformation | |||
- added 50 microL of 5 mg/mL amp to 5 mL LB per culture | |||
- shook @ 180 rpm, 37 dC overnight | |||
==='''06.14.06: Wed.'''=== | |||
====''Miniprep''==== | |||
1. Plasmids (2 samples each) | |||
R0010: lac operon promoter | |||
E7104: T7 promoter + GFP | |||
E0241: GFP | |||
2. Protocol | |||
- w/ 5 mL samples, 1 mL saved for glycerol stock | |||
- rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet | |||
- supernatant removed and pellet resuspended in 250 microL Buffer P1 | |||
- 250 microL Buffer P2 added and tube inverted 4-6 times for mixing | |||
(no vortexing to prevent shearing of genomic DNA; let sit for no longer than 5 min) | |||
- 350 microL Buffer N3 added and tube inverted 4-6 times for mixing | |||
- centrifuged @ 13,000 rpm for 10 min | |||
(formation of white pellet) | |||
- supernatant transferred to QIAprep spin column and centrifuged for 1 min | |||
- 0.5 mL Buffer PB added and centrifuged for 1 min | |||
(optional) | |||
- added 0.75 mL Buffer PE for washing and centrifuged for 1 min | |||
- flowthrough discarded and centrifuged for 1 min to remove residual buffer | |||
- QIAprep column transferred to 1.5 eppendorfs | |||
- 50 microL water added to center of column | |||
(let column stand for 1 min) | |||
- centrifuged for 1 min | |||
- nanodropped | |||
====''Digestion''==== | |||
1. Materials | |||
8 microL DNA (R0010, E0241) | |||
2.5 microL 10x BSA | |||
2.5 microL 10x Buffer (Buffer 2) | |||
11 microL dH2O | |||
0.5 microL enzyme 1 and 2 (SpeI and PstI, XbaI and PstI) | |||
total vol = 25 microL | |||
2. Protocol | |||
- checked www.neb.com to determine correct Buffer | |||
- digested @ 37 dC for 1 hr | |||
- heat shocked @ 97 dc for 15 min to inactivate restriction enzymes | |||
- added 0.1 microL (1 unit) of 10,000 units/mL CIP to vector DNA (R0010) | |||
- incubated vector DNA @ 37 dC for 1 hr | |||
====''Gel Electrophoresis and Purification''==== | |||
1. 1.0% Agarose Gel | |||
L. 1: 1 kb Ladder | |||
L. 2: R0010 Sample 1 | |||
L. 3: R0010 Sample 2 | |||
L. 4: E0241 Sample 1 | |||
L. 5: E0241 Sample 2 | |||
2. Image | |||
[[Image:Pic 2.jpg|thumb|center]] | |||
==='''06.15.06: Thurs.'''=== | |||
====''Ligation''==== | |||
====''Transformation''==== | |||
==Week 2== | |||
==='''06.19.06: Mon.'''=== | |||
====''Presentation: Project Proposal''==== | |||
[[Media:Week 2 Presentation.ogg]] |
Revision as of 12:11, 18 June 2006
Week 1
06.12.06: Mon.
Folding DNA Nanostructures
1. Working Stocks
44 nM scaffold (20 microL) 0.99 microM of each oligo
2. Protocol
- goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 microL - calculations: scaffold = (10 nM)/(44 nM) * 20 microL = 4.5 microL oligos = (100 nM)/(990 nM) * 20 microL = 2 microL - reaction mixture: 4.5 microL p7308 scaffold 2 microL oligos 2 microL 10x folding buffer (500 mM HEPES ph 7.5, 500 mM NaCl, 100 mM MgCl2) 11.5 microL dH2O - annealing times: 90 dC, 5' 65 dC, 20' 55 dC, 20' 45 dC, 20' 37 dC, 30' - neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water
Transformation
1. Plasmids
R0010: lac operon promoter E7104: T7 promoter + GFP E0241: GFP
2. Protocol
- let chemically competent OneShot Top10 cells thaw on ice - added appropriate DNA to cells and tapped gently to mix - let sit on ice for 20 min - heat shocked @ 42 dC for 30" - let cool on ice for 2 min - added 200 microL SOC media - shook @ 37 dC for 1 hr - pipetted and spread onto agar plates treated w/ ? - incubated @ 37 dC overnight agar side-up
06.13.06: Tues.
Gel Electrophoresis w/ 2% Gel
1. Protocol
- mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask - heated for 1.5 min in microwave w/ top loosely screwed on - added 3 microL EtBr - poured into gel frame and let set for 20 min - placed into running box and submerged w/ TBE - wells loaded - ran gel @ 130 V, 45 min
2. Samples
L. 1: 1 kB ladder L. 2 and 3: DNA nanostructure L. 4 and 5: neg control w/ no oligonucleotides L. 6 and 7: neg control w/ no scaffold L. 8: 1 kB ladder loading dye: BTB dye 50% glycerol + 10X TBE ladder: 10 microL + 1 microL dye samples: 10 microL + 1 microL dye
3. Image
Transformation P. 2
1. Protocol
- prepared 2 liquid cultures for each transformation - added 50 microL of 5 mg/mL amp to 5 mL LB per culture - shook @ 180 rpm, 37 dC overnight
06.14.06: Wed.
Miniprep
1. Plasmids (2 samples each)
R0010: lac operon promoter E7104: T7 promoter + GFP E0241: GFP
2. Protocol
- w/ 5 mL samples, 1 mL saved for glycerol stock - rest of samples transferred to 1.5 eppendorfs and centrifuged to form pellet - supernatant removed and pellet resuspended in 250 microL Buffer P1 - 250 microL Buffer P2 added and tube inverted 4-6 times for mixing (no vortexing to prevent shearing of genomic DNA; let sit for no longer than 5 min) - 350 microL Buffer N3 added and tube inverted 4-6 times for mixing - centrifuged @ 13,000 rpm for 10 min (formation of white pellet) - supernatant transferred to QIAprep spin column and centrifuged for 1 min - 0.5 mL Buffer PB added and centrifuged for 1 min (optional) - added 0.75 mL Buffer PE for washing and centrifuged for 1 min - flowthrough discarded and centrifuged for 1 min to remove residual buffer - QIAprep column transferred to 1.5 eppendorfs - 50 microL water added to center of column (let column stand for 1 min) - centrifuged for 1 min - nanodropped
Digestion
1. Materials
8 microL DNA (R0010, E0241) 2.5 microL 10x BSA 2.5 microL 10x Buffer (Buffer 2) 11 microL dH2O 0.5 microL enzyme 1 and 2 (SpeI and PstI, XbaI and PstI) total vol = 25 microL
2. Protocol
- checked www.neb.com to determine correct Buffer - digested @ 37 dC for 1 hr - heat shocked @ 97 dc for 15 min to inactivate restriction enzymes - added 0.1 microL (1 unit) of 10,000 units/mL CIP to vector DNA (R0010) - incubated vector DNA @ 37 dC for 1 hr
Gel Electrophoresis and Purification
1. 1.0% Agarose Gel
L. 1: 1 kb Ladder L. 2: R0010 Sample 1 L. 3: R0010 Sample 2 L. 4: E0241 Sample 1 L. 5: E0241 Sample 2
2. Image