Team:Stanford/Notebook/Lab Work/Week 5

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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)

1. RFP and GFP: cut with EcoRI, SpeI (2 digestions each)
2. B1006 terminator: cut with EcoRI 2 hours, heat kill 80oC 20 minutes, then add XbaI overnight
3. B1006: cut with XbaI and SpeI
4. 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)
    
    

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.