Team:Stanford/Notebook/Lab Work/Week 5
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*vector=B1006 linearized (cut with EcoRI, XbaI; inserts=GFP or RFP cut with EcoRI, SpeI (6 ligations set up) | *vector=B1006 linearized (cut with EcoRI, XbaI; inserts=GFP or RFP cut with EcoRI, SpeI (6 ligations set up) | ||
*Francisco transformed these | *Francisco transformed these | ||
+ | |||
+ | ===Chris's Notebook=== | ||
+ | |||
+ | embCAB Promoter: run diagnostic of restriction digest; if good, Enzymatic | ||
+ | reaction cleanup; elute into 10 ul of MQ h20. | ||
+ | |||
+ | Ligations: | ||
+ | |||
+ | -F2620+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb3C5 | ||
+ | |||
+ | -embCAB+sfGFP+psb3C5 | ||
+ | |||
+ | New Ligation Protocol | ||
+ | |||
+ | (Mix on Ice) | ||
+ | |||
+ | 11 ul of H20 | ||
+ | |||
+ | 2 ul of each digested product | ||
+ | |||
+ | 2 ul of 10X T4 DNA Ligase Reaction Buffer | ||
+ | |||
+ | 1 ul of T4 DNA Ligase | ||
+ | |||
+ | Incubate at RT for 10 min | ||
+ | |||
+ | Incubate 80 C for 20 minutes | ||
+ | |||
+ | Store at -20 C | ||
+ | |||
+ | Transform Ligations | ||
==7/28 Wednesday== | ==7/28 Wednesday== | ||
Line 155: | Line 190: | ||
*pBAD (I0500) | *pBAD (I0500) | ||
*pLUX (F2620) | *pLUX (F2620) | ||
+ | |||
+ | ===Chris's Notebook=== | ||
+ | |||
+ | embCAB: restriction digest was successful; now have 4 X 10 ul of purified, | ||
+ | digested embCAB promoter ready for ligation | ||
+ | |||
+ | Ligations and Transformations | ||
+ | |||
+ | Only -F2620+sfGFP+psb1A2 and the +3C5 control grew; nothing else, not even the | ||
+ | +1A2 control grew—maybe the newer ampicillin plates needed time in the cold room. | ||
+ | |||
+ | Re-transform: | ||
+ | |||
+ | -F2620+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb3C5 | ||
+ | |||
+ | +1A2 | ||
+ | |||
+ | Re-ligate using aliquoted T4 DNA Ligase Buffer: | ||
+ | |||
+ | -F2620+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb3C5 | ||
+ | |||
+ | -embCAB+sfGFP+psb3C5 | ||
+ | |||
+ | -embCAB+sfGFP+psb1A2 | ||
+ | |||
+ | New Ligation Protocol | ||
+ | |||
+ | (Mix on Ice) | ||
+ | |||
+ | 11 ul of H20 | ||
+ | |||
+ | 2 ul of each digested product | ||
+ | |||
+ | 2 ul of 10X T4 DNA Ligase Reaction Buffer | ||
+ | |||
+ | 1 ul of T4 DNA Ligase | ||
+ | |||
+ | Incubate at RT for 10 min | ||
+ | |||
+ | Incubate 80 C for 20 minutes | ||
+ | |||
+ | Store at -20 Ce | ||
+ | |||
+ | Restriction Digests | ||
+ | |||
+ | pBAD, 3C5, sfGFP, 1A2, F2620 | ||
+ | |||
+ | Plasmid | ||
+ | |||
+ | Each enzyme | ||
+ | |||
+ | NEB buffer 2 | ||
+ | |||
+ | 10x BSA | ||
+ | |||
+ | NP water | ||
+ | |||
+ | Inoculated ccdb cultures | ||
+ | |||
+ | 12.0 μl | ||
+ | |||
+ | 1.0 μl | ||
+ | |||
+ | 5.0 μl | ||
+ | |||
+ | 5 μl (optional depending on enzymes used) | ||
+ | |||
+ | Bring to 50 μl total volume (26 ul) | ||
==7/29 Thursday== | ==7/29 Thursday== | ||
Line 231: | Line 342: | ||
Twitter slide: change title to Gold Medal Requirements (add Facebook?) | Twitter slide: change title to Gold Medal Requirements (add Facebook?) | ||
Lab Logistics: suggestions to collaborate in lab more efficiently | Lab Logistics: suggestions to collaborate in lab more efficiently | ||
+ | |||
+ | ===Chris's Notebook=== | ||
+ | |||
+ | Miniprep Plasmid Backbones for 3A/ccdb Free Assembly | ||
+ | |||
+ | Free Assembly of: | ||
+ | |||
+ | -F2620+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb1A2 | ||
+ | |||
+ | -pBAD+sfGFP+psb3C5 | ||
+ | |||
+ | -embCAB+sfGFP+psb3C5 | ||
+ | |||
+ | -embCAB+sfGFP+psb1A2 | ||
==7/30 Friday== | ==7/30 Friday== | ||
+ | ===Francisco's Notebook=== | ||
+ | Starting afresh with Christina’s help. | ||
+ | |||
+ | '''GFP, RFP - terminator ligations:''' | ||
+ | |||
+ | Inoculated GFP, RFP, and Term B1006. | ||
+ | * 2 glass tubes each, 6 mL of LB in each glass tube. | ||
+ | |||
===Alex's Notebook=== | ===Alex's Notebook=== | ||
Line 299: | Line 434: | ||
**record audio (in as many languages as possible) | **record audio (in as many languages as possible) | ||
+ | ===Chris's Notebook=== | ||
+ | |||
+ | 3A Assembly of: | ||
+ | |||
+ | -F2620+sfGFP+psb4C5 | ||
+ | -I746908+psb4C5 (this part was only characterized in psb1A2; meeting a gold medal | ||
+ | requirement) | ||
+ | -embCAB+sfGFP+psb4C5 (to determine baseline noise) | ||
+ | J23100+sfGFP+psb4C5 (maybe as a new standard for classifying constitutive | ||
+ | promoters) | ||
+ | J23100+B0034+psb4C5 (first step of cloning scheme) | ||
+ | |||
+ | Got approximately 1-3 colonies per plate-need to increase insert:vector. | ||
+ | Also do PCR purification of Restriction Digests elute in 30 ul | ||
+ | |||
+ | http://bioinfo.clontech.com/infusion/molarRatio.do | ||
+ | |||
+ | psb4C5: 3200 | ||
+ | F2620 (1000):30 ng insert: | ||
+ | sfGFP (800): 75 ng insert: | ||
+ | embCAB (250): 50 ng insert | ||
+ | J23100 (35): 50 ng insert: | ||
+ | B0034 (12): 50 ng insert: | ||
+ | |||
+ | F2620+sfGFP+psb4C5: 3 ul of F2620, 7.5 ul of sfGFP, 1 ul of psb4C5, 5.5 of mq | ||
+ | |||
+ | I746908+psb4C5: 1 ul of psb4C5, 2 ul of I746908 14 of mq | ||
+ | |||
+ | -embCAB+sfGFP+psb4C5: 5 ul of pemb, 7.5 ul of sGFP, 1 ul of psb4C5, 3.5 of mq | ||
+ | |||
+ | J23100+sfGFP+psb4C5 : 5 ul of 100, 1 ul of psb4C5, 7.5 ul of sfGFP, 3.5 of mq | ||
+ | |||
+ | J23100+B0034+psb4C5: 5 ul of 100, 1 ul of psb4C5, 5 ul of B0034, 6 ul of mq | ||
+ | |||
+ | ==7/31 Saturday== | ||
+ | ===Francisco's Notebook=== | ||
+ | '''GFP, RFP - terminator ligations:''' | ||
+ | *Miniprepped GFP, RFP and Term B1006 | ||
+ | *Used Promega miniprep kit. Eluted with 50 mL water (?) for each of the 2 microfuge tube. Combined volumes into one tube. | ||
+ | |||
+ | ! Tip: if planning on making freezer stocks, save some liquid culture before miniprep. | ||
+ | |||
+ | ! Tip: use one microfuge tube per glass tube. Decant culture into microfuge tube, spin down, decant supernatant, and repeat. This gets all the cells into one pellet in one microfuge tube and saves tubes. | ||
+ | |||
+ | ! Tip: vortex before lysing cells to thoroughly resuspend cells. Do not vortex after lysing, but invert tube with some force to mix. | ||
+ | |||
+ | Miniprep Gel Diagnostic | ||
+ | *Used unknown percent gel, 95V, ~30 minutes. DNA concentration estimates from bands: | ||
+ | *GFP: ~ 50 ng/uL | ||
+ | *RFP: ~75-100 ng/uL | ||
+ | *Term: ~50 ng/uL | ||
+ | |||
+ | ! Tip: Set up two 15-well combs in a diagnostic gel, run the lower lanes first and then the upper lanes to get the most out of a gel. | ||
+ | |||
+ | Digestions of GFP, RFP and Term | ||
+ | *Based on the estimated, and a target of 2 mg of DNA in a digestion, we set up these digestion recipes: | ||
+ | *Term: 34 uL DNA, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL XbaI* | ||
+ | *GFP: 34 uL DNA, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL SpeI* | ||
+ | *RFP: 25 uL DNA, 9 uL water, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL SpeI* | ||
+ | *Do digestions sequentially, add first enzyme, heat inactivate, take 1 uL sample for diagnostic gel later, and add second enzyme. | ||
+ | *First enzyme added 1pm, heat inactivated 9:15pm | ||
+ | *Second enzyme ran overnight. | ||
+ | |||
+ | Freezer Stock of GFP, RFP, Term B1006 | ||
+ | *Back dilute 350 uL of liquid culture into 5 mL of LB and incubate in shaker for around 4 hours (until cells are in late exponential phase). | ||
+ | *Add 0.5 mL of glycerol and 1.5 mL of culture to a cryovial, invert a bunch of times, and store in the -80 deg C freezer. | ||
+ | *Use sterile technique throughout. | ||
+ | |||
+ | '''Promoter - RSID, sRNA ligations''' | ||
+ | *Miniprep Gel Diagnostic of pLux (F2620) | ||
+ | *Miniprepped by Laura earlier | ||
+ | *estimated pLux concentration: 50 ng/uL | ||
+ | |||
+ | ===Karina's Notebook=== | ||
+ | '''Make Freezer Stock from innoculations'''<br/> | ||
+ | The technique is called "Back dilution"<br/> | ||
+ | *Get new test tubes for liquid culture | ||
+ | *add 5 mL media + 5 mL cell sample | ||
+ | *put back in shaker, leave for a few hours<br/><br/> | ||
+ | |||
+ | '''Miniprep'''<br/> | ||
+ | *Use 2 tubes for each cell tube | ||
+ | **to concentrate the 2 tubes, add sample to microcentrifuge tubes, spin, dump out supernatant and add more sample | ||
+ | *before resuspension spin, asparate any extra media in tubes. The extra media can mess up digestions. | ||
+ | *When resuspending, vortex cells to make sure that the pellet is in solution | ||
+ | *When adding buffers, work quickly and invert with force <br/><br/> | ||
+ | == Sunday August 1 == | ||
+ | ===Francisco's Notebook=== | ||
+ | GFP, RFP - terminator ligations | ||
+ | Digestion Diagnostic Gel | ||
+ | *0.8% w/v agarose gel | ||
+ | *Bands for terminator, RFP, and GFP look good. First digestion of GFP (EcoRI) was slightly incomplete. | ||
+ | *Estimated digested DNA concentrations: | ||
+ | **RFP/GFP: 30-40 ng/uL | ||
+ | **Term: ~30 ng/uL | ||
- | < | + | ! Tip: If taking samples from water bath, quickspin to get condensation down. If taking samples from the freezer, thaw, flick to mix, and quickspin to get all liquid down. |
+ | ! Tip: Scrunch kimwipe to stopper gel flask. | ||
+ | ! Tip: < 1 min microwave time should be sufficient. | ||
+ | ! Tip: When loading, set up 4 uL beads of water on parafilm first, then add 1 uL dye, then add sample -- minimizes pipette tips used. | ||
+ | ! Tip: For gels of digestions, run uncut, singly cut and doubly cut next to each other. |
Latest revision as of 00:50, 27 October 2010
![](http://openwetware.org/images/8/8d/Quad_center.jpg)
Home | Project | Applications | Modeling | Parts | Team | Notebook |
Spring: Brainstorming | Spring Meetings
Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries
|
7/26 Monday
Alex's Notebook
Get picture from gel imager. Run the gel. Gel extract. Ligate. Transform. Plate. Both BW and DH5alpha. Order: Heat shock cells EcoRI-HF T4 Ligase MinElute tubes (?)
PCR:
RBS’s
100 ng template
.5 ul primers (200 nM). Also try 2 (800 nM).
Promoters Try varying concentrations for NC and C 20 uM primer solution, 50 ul tot. vol. Want: 100, 200, 300, 400, 500 nM conc. Use: .25, .5, .75, 1, and 1.25 uL primers and 4.5, 4, 3.5, 3, and 2.5 uL H2O. Try 200, 400, 1000 (2.5 uL), and 2000 (5 uL) first
Try 2+3, then +1+4, one PCR purified and other not. 20 uM primer solution, 50 ul tot. vol.
40 sm, 2+3, PCR purify, transfer. 5 uL each primer. SHOULD NOT WORK!!! 40 sm, 2+3, direct transfer. 40 sm, 1+4+2+3. 4 uL primer 1, 4 uL primer 4, and 2 of template (transfer). 40 sm, 1+4+2+3. 4 uL primer 1, 4 uL primer 4, and 2 of template (transfer).
45 sm, 2+3, PCR purify, transfer. 2.5 uL each primer. 45 sm, 2+3, direct transfer. 45 sm, 1+4+2+3. 2 uL primer 1, 2 uL primer 4, and 1 of template (transfer). 45 sm, 1+4+2+3. 2 uL primer 1, 2 uL primer 4, and 1 of template (transfer).
From best of these reactions, take .5 of P1 and P4 with .1 PCR purified template. Use YA, 30 cycles.
Laura's Notebook
helped Francisco aliquot competent cells (thaw main tube- 1 mL- on ice, transfer 50 uL to each of 20 pre-chilled 0.5 mL microfuge tubes)
7/27 Tuesday
Alex's Notebook
Continue w/ PCR. 100 seems okay. Nothing from 107, since sequence is messed up. I5 and F26 look ok. I0500 (1255 bp) Good F2620 (1106 bp) Good enough.
J100 (98 vs 62 bp) Good enough. J107 (98 bp) Nothing.
AfsS C (123 bp) Good AfsS NC (104 bp) Good
Next steps: PCR purify (regular and MinElute). Nanodrop. RD: E/P. Spacer should be enough. Heat inactivate (80 C for 20 min, 75 C for 20 min for Klenow), RD cleanup. Nanodrop. Ligate. Ratios: 20 uL total: 1 uL ligase, 2 uL buffer, 8 uL, 8 uL insert, 375 ng vector. Heat inactivate (65 C for 20 min.) and transform.
New scheme for AfsS assembly: 1+2, 3+4, gel electrophoresis to get rid of primers. Use -------------- for primer concentration. Gel extract wanted product. Mix in a single reaction. Use same as above.
Check plates. Some grew. Culture O/N and do colony PCR. Focus on parts needed. RD today. All parts. Get extra RD enzyme (BglI). Control: 1A2 @ EcoRI, and use blunting enzyme from Ryan. 100 uL total: 24 uL DNA, 52 uL H2O, 10 uL BSA, 10 uL NEB buffer (check which to use), 4 uL enzymes. Gel electrophoresis and extract. Ligate and heat inactivate (as above). Transform. Use brown stripe or use new ligase buffer. NEED MORE LIGASE!!! 1.5 uL DNA, incubate on ice.
Prepare more electro-competent cells. Use DH5 alpha 21. Check for space in the incubator.
Laura's Notebook
redo failed ligation: vary vector/insert ratios
prior recipe | ligation 1 | ligation 2 | ligation 3 | |
water | none | 11.0 | 7.0 | none |
vector | 5.0 | 2.0 | 2.0 | 2.0 |
insert | 12.0 | 4.0 | 8.0 | 15.0 |
10X buffer | 2.0 | 2.0 | 2.0 | 2.0 |
ligase | 1.0 | 1.0 | 1.0 | 1.0 |
- vector=B1006 linearized (cut with EcoRI, XbaI; inserts=GFP or RFP cut with EcoRI, SpeI (6 ligations set up)
- Francisco transformed these
Chris's Notebook
embCAB Promoter: run diagnostic of restriction digest; if good, Enzymatic reaction cleanup; elute into 10 ul of MQ h20.
Ligations:
-F2620+sfGFP+psb1A2
-pBAD+sfGFP+psb1A2
-pBAD+sfGFP+psb3C5
-embCAB+sfGFP+psb3C5
New Ligation Protocol
(Mix on Ice)
11 ul of H20
2 ul of each digested product
2 ul of 10X T4 DNA Ligase Reaction Buffer
1 ul of T4 DNA Ligase
Incubate at RT for 10 min
Incubate 80 C for 20 minutes
Store at -20 C
Transform Ligations
7/28 Wednesday
Alex's Notebook
Cultivate cells. PCR 1,2 and 3,4. Gel, gel extract.
Laura's Lab Notebook
miniprepped (ProMega kit): GFP and B1006 terminator (RFP didn't grow) NanoDrop data:
sample | 260/280 | 260/230 | ng/uL |
B1006 | 1.61 | 0.95 | 49.4 |
GFP | 1.83 | 1.22 | 96.4 |
ran 2% diagnostic gel, 75V, 1 hour
- order: 100bp ladder, RSID1C PCR product, GFP digestion, RFP digestion, terminator digestion
- all samples: 1 uL sample + 1 uL loading dye
ran 2% gel for gel extraction, 75V, 1 hour
- order: 100bp ladder, GFP digestion, RFP digestion
- 10 uL loading dye added to 50 uL digestions, 40 uL loaded on gel
gel extraction of GFP, RFP digestion products (Qiagen kit)
tube (g) | tube + slice (g) | gel slice (g) | uL QG | |
GFP | 1.01 | 1.03 | 0.02 | 60 |
RFP | 1.01 | 1.03 | 0.02 | 60 |
ran 2% diagnostic gel of gel purified GFP and RFP digests
- order: 100bp ladder, GFP, RFP
- 1 uL sample + 1 uL dye + 4 uL H2O
B1006 terminator NanoDrop data (diluted 50:50 with H2O)
260/280 | 260/230 | ng/uL |
1.47 | 1.39 | 10.4 |
- = 20.8 ng/uL in original sample
Digest concentrations still very low... set up larger overnight cultures- 9 mL cultures, 2 cultures each part:
- B1006 (terminator)
- GFP
- RFP
- pBAD (I0500)
- pLUX (F2620)
Chris's Notebook
embCAB: restriction digest was successful; now have 4 X 10 ul of purified, digested embCAB promoter ready for ligation
Ligations and Transformations
Only -F2620+sfGFP+psb1A2 and the +3C5 control grew; nothing else, not even the +1A2 control grew—maybe the newer ampicillin plates needed time in the cold room.
Re-transform:
-F2620+sfGFP+psb1A2
-pBAD+sfGFP+psb1A2
-pBAD+sfGFP+psb3C5
+1A2
Re-ligate using aliquoted T4 DNA Ligase Buffer:
-F2620+sfGFP+psb1A2
-pBAD+sfGFP+psb1A2
-pBAD+sfGFP+psb3C5
-embCAB+sfGFP+psb3C5
-embCAB+sfGFP+psb1A2
New Ligation Protocol
(Mix on Ice)
11 ul of H20
2 ul of each digested product
2 ul of 10X T4 DNA Ligase Reaction Buffer
1 ul of T4 DNA Ligase
Incubate at RT for 10 min
Incubate 80 C for 20 minutes
Store at -20 Ce
Restriction Digests
pBAD, 3C5, sfGFP, 1A2, F2620
Plasmid
Each enzyme
NEB buffer 2
10x BSA
NP water
Inoculated ccdb cultures
12.0 μl
1.0 μl
5.0 μl
5 μl (optional depending on enzymes used)
Bring to 50 μl total volume (26 ul)
7/29 Thursday
Alex's Notebook
Transform using ccdb vector w/ new ligase and ligase buffer. Final PCR assembly 1,2+3,4. Failure.
Karina's Notebook
Goal: We want to try BioBrick 3A Free Assembly! [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf Protocol found here.]
Miniprep DNA
Helped Chris miniprep plasmids that contain ccdb to use for 3A assembly. Used [http://www.promega.com/tbs/tb225/tb225.pdf Wizard Plus Protocol] (note: Procedure takes the whole morning!)
Forgot to concentrate cells at resuspension stage (crap) so concentrated them at centrifuagtion stage. Akin to the way I do so for PCR cleanup.
Nanodrop Results
Concentration (ng/uL) | 260/280 | 260/230 | |
4K5 | 100.2 | 1.69 | 0.84 |
4K5 | 80.9 | 1.70 | |
4K5 | 97.3 | 1.85 | 1.22 |
J64100 | 45.4 | 1.81 | 0.89 |
J64100 | 58.5 | 1.86 | 1.06 |
J64100 | 69.0 | 1.79 | 0.93 |
Laura's Notebook
Miniprepped (ProMega kit): RFP, GFP, B1006 terminator, F2620 (pBAD didn't grow)
- all 18 mL (2- 9mL cultures) combined into one miniprep for each
- Francisco did nanodrop: he has concentration data
set up digestions for both types of ligations (two-part and 3a protocol)
- RFP and GFP: cut with EcoRI, SpeI (2 digestions each)
- B1006 terminator: cut with EcoRI 2 hours, heat kill 80oC 20 minutes, then add XbaI overnight
- B1006: cut with XbaI and SpeI
- 4C5 backbone: cut with EcoRI, PstI
- for two part ligations: use GFP or RFP from #1, terminator from #2 (2 ligations- one for GFP, one for RFP)
- for 3a ligation: use GFP or RFP from #1, terminator from #3, 4C5 backbone from #4 (2 ligations- one for GFP, one for RFP)
Meeting with Rayka- suggestions for Friday's (tomorrow's) meeting:
- include a brief review/overview of project for clarification
- basic schematics of the two sides of the project
- details of each, including the devices we plan to build
- ligation schemes in visuals
- for digital, analog: 1. overall plan 2. ligation scheme (home slide) 3. troubleshooting/challenges (including potential solutions)
suggestion for ligations: add ATP (is most likely to go bad in buffer) include plans for next week: minimum and ideal agenda again after lab stuff Twitter slide: change title to Gold Medal Requirements (add Facebook?) Lab Logistics: suggestions to collaborate in lab more efficiently
Chris's Notebook
Miniprep Plasmid Backbones for 3A/ccdb Free Assembly
Free Assembly of:
-F2620+sfGFP+psb1A2
-pBAD+sfGFP+psb1A2
-pBAD+sfGFP+psb3C5
-embCAB+sfGFP+psb3C5
-embCAB+sfGFP+psb1A2
7/30 Friday
Francisco's Notebook
Starting afresh with Christina’s help.
GFP, RFP - terminator ligations:
Inoculated GFP, RFP, and Term B1006.
- 2 glass tubes each, 6 mL of LB in each glass tube.
Alex's Notebook
Colony PCR: 1. sG 2. FsG 3. I5E 4. B34 + J100. 1A2: 78 bp + 120 bp = 198 bp 3C5: 115 bp + = bp
Diagnostic of PCR and RD products. RD: 3 samples, ~350 ng/uL 1. B34 2. J100 3. J119
PCR: 6 samples, ~48, ~50, ~48, ~16, ~42, ~32. 1. C 2. C 3. NC 4. F26 5. I5 6. J100
Karina's Notebook
Friday Lab Meeting:
Troubleshooting
- Ligations not working
- Put in enough DNA in mass
- AT LEAST 200 ng, if below 200 ng, its not worth doing a ligation reaction
- cells may not be electrocompetent enough
- spike ATP to ligation buffer?
- 1 uL of 1mM ATO for 10 uL ligation reaction
- sRNA_C PCR assembly
- retrace what we ordered and check concentration of primers (may not be enough)
- retrace what we ordered and check concentration of primers (may not be enough)
Flow Cytometry Info
- Introductory training sessions take a couple of months (eek!)
- can't get access everyday of the week, so don't depend on it
- Scanford machine: an older maching that only reads GFP, less popular, could probably get access to this one more easily!
- Kathy Crumpton: technician we need to talk to, need to check that the Scanford can read E. coli
Laura's Notebook
9am iGEM meeting
in attendance: team (Chris, Karina, Alex, Francisco, Laura), Christina, Ryan, Rayka, Anusuya, Graham
Things to accomplish/address by next week:
- potential names of circuits (Laura, Rayka)
- flow cytometry: Does Scanford work for E. Coli? (Chris, Graham)
- get ligations to work! (everyone)
- make sure DNA concentration is high enough
- when looking at gel to estimate concentration, look directly at gel (not at image on computer screen)
- use fresh ligase buffer (or add ATP)
- get successful ligation to use as positive control (from Ryan?)
- get RSID1C PCR working
- check concentration of primers: mix well, make new working stock
- double check annealing temperatures, correct sequences
- 4 piece PCR (Alex)- order as 2 pieces?
- circuit diagrams, including levels of abstraction, consistent formats
- Twitter:
- list/group
- record audio (in as many languages as possible)
Chris's Notebook
3A Assembly of:
-F2620+sfGFP+psb4C5 -I746908+psb4C5 (this part was only characterized in psb1A2; meeting a gold medal requirement) -embCAB+sfGFP+psb4C5 (to determine baseline noise) J23100+sfGFP+psb4C5 (maybe as a new standard for classifying constitutive promoters) J23100+B0034+psb4C5 (first step of cloning scheme)
Got approximately 1-3 colonies per plate-need to increase insert:vector. Also do PCR purification of Restriction Digests elute in 30 ul
http://bioinfo.clontech.com/infusion/molarRatio.do
psb4C5: 3200 F2620 (1000):30 ng insert: sfGFP (800): 75 ng insert: embCAB (250): 50 ng insert J23100 (35): 50 ng insert: B0034 (12): 50 ng insert:
F2620+sfGFP+psb4C5: 3 ul of F2620, 7.5 ul of sfGFP, 1 ul of psb4C5, 5.5 of mq
I746908+psb4C5: 1 ul of psb4C5, 2 ul of I746908 14 of mq
-embCAB+sfGFP+psb4C5: 5 ul of pemb, 7.5 ul of sGFP, 1 ul of psb4C5, 3.5 of mq
J23100+sfGFP+psb4C5 : 5 ul of 100, 1 ul of psb4C5, 7.5 ul of sfGFP, 3.5 of mq
J23100+B0034+psb4C5: 5 ul of 100, 1 ul of psb4C5, 5 ul of B0034, 6 ul of mq
7/31 Saturday
Francisco's Notebook
GFP, RFP - terminator ligations:
- Miniprepped GFP, RFP and Term B1006
- Used Promega miniprep kit. Eluted with 50 mL water (?) for each of the 2 microfuge tube. Combined volumes into one tube.
! Tip: if planning on making freezer stocks, save some liquid culture before miniprep.
! Tip: use one microfuge tube per glass tube. Decant culture into microfuge tube, spin down, decant supernatant, and repeat. This gets all the cells into one pellet in one microfuge tube and saves tubes.
! Tip: vortex before lysing cells to thoroughly resuspend cells. Do not vortex after lysing, but invert tube with some force to mix.
Miniprep Gel Diagnostic
- Used unknown percent gel, 95V, ~30 minutes. DNA concentration estimates from bands:
- GFP: ~ 50 ng/uL
- RFP: ~75-100 ng/uL
- Term: ~50 ng/uL
! Tip: Set up two 15-well combs in a diagnostic gel, run the lower lanes first and then the upper lanes to get the most out of a gel.
Digestions of GFP, RFP and Term
- Based on the estimated, and a target of 2 mg of DNA in a digestion, we set up these digestion recipes:
- Term: 34 uL DNA, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL XbaI*
- GFP: 34 uL DNA, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL SpeI*
- RFP: 25 uL DNA, 9 uL water, 5 uL NEBuffer 4, 5 uL 10x BSA, 3 uL EcoRI, 3 uL SpeI*
- Do digestions sequentially, add first enzyme, heat inactivate, take 1 uL sample for diagnostic gel later, and add second enzyme.
- First enzyme added 1pm, heat inactivated 9:15pm
- Second enzyme ran overnight.
Freezer Stock of GFP, RFP, Term B1006
- Back dilute 350 uL of liquid culture into 5 mL of LB and incubate in shaker for around 4 hours (until cells are in late exponential phase).
- Add 0.5 mL of glycerol and 1.5 mL of culture to a cryovial, invert a bunch of times, and store in the -80 deg C freezer.
- Use sterile technique throughout.
Promoter - RSID, sRNA ligations
- Miniprep Gel Diagnostic of pLux (F2620)
- Miniprepped by Laura earlier
- estimated pLux concentration: 50 ng/uL
Karina's Notebook
Make Freezer Stock from innoculations
The technique is called "Back dilution"
- Get new test tubes for liquid culture
- add 5 mL media + 5 mL cell sample
- put back in shaker, leave for a few hours
Miniprep
- Use 2 tubes for each cell tube
- to concentrate the 2 tubes, add sample to microcentrifuge tubes, spin, dump out supernatant and add more sample
- before resuspension spin, asparate any extra media in tubes. The extra media can mess up digestions.
- When resuspending, vortex cells to make sure that the pellet is in solution
- When adding buffers, work quickly and invert with force
Sunday August 1
Francisco's Notebook
GFP, RFP - terminator ligations Digestion Diagnostic Gel
- 0.8% w/v agarose gel
- Bands for terminator, RFP, and GFP look good. First digestion of GFP (EcoRI) was slightly incomplete.
- Estimated digested DNA concentrations:
- RFP/GFP: 30-40 ng/uL
- Term: ~30 ng/uL
! Tip: If taking samples from water bath, quickspin to get condensation down. If taking samples from the freezer, thaw, flick to mix, and quickspin to get all liquid down. ! Tip: Scrunch kimwipe to stopper gel flask. ! Tip: < 1 min microwave time should be sufficient. ! Tip: When loading, set up 4 uL beads of water on parafilm first, then add 1 uL dye, then add sample -- minimizes pipette tips used.
! Tip: For gels of digestions, run uncut, singly cut and doubly cut next to each other.