Team:UC-Santa Cruz/Notebook2

Protocols

1. Make electrocompetent CB15N (for immediate use)

         0. Grow colony pick overnight CB15N in PYE @ 37Cº

1. Check OD600 of culture

2. Balance culture tubes < 0.01 g difference

3. Spin at 3000 rpm 4Cº for 15 minutes

4. Discard supernatant

5. Add 10 mL Milli Q water

6. Spin at 3000 rpm 4Cº for 15 minutes

7. Discard supernatant.

8. Repeat step 5-7 three times

           9. Make aliquots and put in deep freezer.

2. Miniprep on E. coli cultures

         1.Spin E. coli culture at 3000rpm for 5 minutes

         2. Discard supernatant.

         3. Resuspend in 6mL of Milli Q water.

         4. Add 1mL 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes

         5. Add entire mix to blue zymo-midi-filter and  column, place  in 50mL conical tube. spin 5000rcf for 6 minutes

         6.Remove and discard filter and place column on collection tube. Place in benchtop centrifuge at max speed for 30 seconds.

         7. Add 400uL endo-wash and spin at max speed on benchtop centrifuge

         8. Add 400uL Zippy wash buffer , centrifuge at max speed for 1 minute, repeat once.

         9. Transfer to 1.5 mL Eppendorf tube add 150uL water, let sit for 1 minute before spinning at max speed for 1 minute

         10. Check concentration with nanodrop

        

3. Transfect CB15N with plasmids to make two strains of transformed CB15N.

4. Grow strains on plates.

5. Pick colonies for overnights.

6. Induce fluorescence.

7. Check for fluorescence.

Gentamicin Dilution

1. Weigh out .1g Gentamicin powder

2. Place in 50mL microcentrifuge tube

3. Add 10 mL Milli Q water

4. Pull plunger out of 60mL BD syringe

5. Screw on filter tip.

6. Fill syringe with solution

7. Empty syringe into new 50mL microcentifuge tube.

Sterilized colony pick

0. Autoclave growth media in container. (allow liberal space in container for aeration of culture), after cooling, add appropriate amount of antibiotic.

1. Heat tungsten loop on bunsen burner until red hot.

2. Let cool beside flame

3. Dip loop in agarose to check temperature

4. Beneath flame, carefully scrape-off one colony from plate making sure not to get any other colonies on loop.

5. Beneath flame, stick loop with colony in growth media making sure not to touch the sides of the container with loop. swirl around until colony washes off of loop.

6. Incubate in appropriate conditions.

Week 1

8-1-13

• Made and autoclaved PYE
• Prepared another batch of  electrocompetent caulobacter (culture OD600 = 0.615A)
• Induced KAN resistant with xylose transformed caulobacter @ OD600 = 0.664A
• Non induced : 3.25ml PXYFPC-2 + CB15N, 20.5ul antibiotic, 200ml PYE
• Induced: 3.25ml PXYFPC-2+ CB15N, 20.5 ul antibiotic, 200ml PYE, .4g xylose .
• Electroporation of CB15N with PVCFPC-4 .
• Plated on PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two negative controls (see 8-5-13))
• Plated CB15N on KAN plate for negative control. (note : this was not prepared following the electroporation protocol with water instead of plasmid. This was just a drop from a colony pick shaking incubator pick of CB15N )

8-2-13

• Checked fluorescence of PCXFPC-2 : UI (OD600= .76 flouereced, I OD600 = .72 no fluorescence.)

Week 2

8-5-13

• Checked plates
• Negative control (CB15N+KAN electroporated with water): one colony
• Negative control (CB15N+KAN straight from culture): many colonies
• 10uL, 50uL, and 100uL many colonies
• Result: Plates deemed unusable
• Made new PYE + Gentamicin plates

8-6-13

• PXYFPC-2 +CB15N culture to be induced made

         -5mL PXYFPC-2 + CB15N (OD600 = 0.72)

• -20uL KAN
• 50 uL PCR of  stpx and pflI on CB15N
• Gel Electrophoresis on PCR reactions

8-7-13

• Transformation of CB15N with PCXFPC-2 (electroporation) time constant = 5.6
• Transformation of CB15N with PVCFPC-4 (electroporation) time constant = 5.4
• PXYFPC-2 +CB15N culture to be induced made

         -5ml PXYFPC-2 + CB15N (OD600 = 0.72)

         -20ul KAN

·       TD PCR for Stpx and pflI tags from caulobacter, two 15 cycle sets

·       Initial denature of 95 Cº

denature	anneal	elongation	denature	anneal	elongation
95 Cº	56 Cº	68 Cº	95 Cº	49 Cº	68 Cº
:10	:15	2:20	:10	:15	2:20

Week 3

8-13-13

• TD PCR for Stpx and pflI #3, 4, and 5. Two sets of 15 cycles with initial denature at 95 C for 1:00 for #3 and 94 C for 3:00 for #4 and #5

#3

denature	anneal	elongation	denature	anneal	elongation
95 Cº	56 Cº	68 Cº	95 Cº	49 Cº	68 Cº
:10	:15	2:20	:15	:15	2:20

#4

denature	anneal	elongation	denature	anneal	elongation
94 Cº	56 Cº	68 Cº	94 Cº	49 Cº	68 Cº
:15	:30	2:20	:15	:30	2:20

#5

denature	anneal	elongation	denature	anneal	elongation
94 Cº	59 Cº	68 Cº	94 Cº	52 Cº	68 Cº
:15	:15	2:30	:15	:15	2:30

8-15-13

• Made LB
• Checked plates

8-19-13

Gentamicin dilution (10mg/mL)

1. Weigh out 0.1g Gentamicin powder

2. Place in 50mL microcentrifuge tube

3. Add 10 mL Milli q water

4. Pull plunger out of 60mL BD syringe

5. Screw on filter tip

6. Fill syringe with solution

7. Empty syringe into new 50mL microcentrifuge tube.

8.aliquot 100uL, 10uL, 500uL, and 10uL into 1.5ml Eppendorf tubes

• Sterilized inoculation of LB with E. coli + PXYFPC-2 +KAN (30ug/mL final concentration)
• Sterilized inoculation of LB with E. coli + PVCFPC-4 +GENT (15ug/mL final concentration)

8-20-13

Created Glycerol stock of E. coli cultures containing PVCFPC-4

Plasmid Mini-prep of E. coli cultures

·       Culture #1 OD550 .916

·       Culture #2 OD5050 2.149

	Nucleic Acid Concentration	A260	A280	260/280	260/230
Culture #1	21.0 ng/uL	.419	.221	1.89	1.68
Culture #2	299.2 ng/uL	5.983	3.184	1.88	2.13

Week 5

9-?-13

• Use Geneious to design primers (forward and reverse) to get Halorhodopsin (HsHR) out of H.Salinarum.
• Order primers.

9-3-13

• Take chip of frozen glycerol stock H.Salinarum and suspend in Milli Q Water

9-4-13

25 uL PCR using HsHR primers ,HS (template), and GC master mix.

• Negative control without primers labeled (-1)
• Negative control without HS (template) labeled (-2)
• 98Cº -2mins/98Cº 10s 57.2Cº 72Cº 2 min/72Cº 3 min hold  4Cº

9-5-13

• Sterilized inoculation of SOC  with iGEM part BBa_K9000 “9000” + E. coli
• Sterilized inoculation of SOC with iGEM part BBa_K9001 “9001” + E. coli
• Sterilized inoculation of SOC with iGEM part BBa_K9010 “9010” + E. coli
• Made chloramphenicol (1ug/1mL) plates

PCR of Halorhodopsin from Halobacterium Salinarum 30 cycles

25 uL reaction with 2x Phusion GC Master Mix

initial denature	denature	anneal	extension	final extension
98 Cº	98 Cº	57 Cº	72 Cº	72 Cº
2:00	00:10	00:20	1:00	5:00

9-6-13

Plasmid miniprep on 9000, 9010, and 9010 cultures.

         Results:

                  -9000 = 99.2 ng/ml

                  -9001 =99.7 ng/ml

                  -9010 = 38.1 ng/ml

         Gel legend : 1Kb ladder , 9010, 9001, 9000

• Design and order sequencing primers for iGEM parts. same forward and reverse for all three.

Week 6

9-9-13

• Send plasmids with sequencing primers out for sequencing.
• Design and order primers for plasmid backbone.
• 20uL PCR on HS for HsHR
• Run diagnostic gel

         - Gel legend : 1Kb ladder , H? , +, -1, -2,

9-10-13

• 50 ul HsHR PCR labeled “50”
• 98C 2min/ 98Cº :10s/57.2Cº 72Cº 1min/72min/Hold 4Cº

9-11-13

Purification gel

         -Gel Legend: 1Kb ladder, 50

• 50ul HsHR PCR (two reactions “50(1)” and “50(2)”) one negative control with no template “50 -”

Gel Legend : 1Kb ladder , 50(1), 50(2), 50-

9-12-13

50uL PCR to obtain plasmid backbone using 9010 (30.4ng/ul) as template.

initial denature	denature	anneal	extension	final extension
98 Cº	98 Cº	52.2 Cº	72 Cº	72 Cº
:30	:10	:30	1:00	5:00

Gel Legend : 1Kb ladder , 50(1), 50(2), 50-

Two 20uL PCR reactions using part 9010 with different annealing temperatures. One at 55 Cº and one at 57 Cº

Gel Legend : 1Kb ladder , 9010(55 Cº) , 9010 neg (55 Cº), 9010 (57 Cº), 9010 neg (57Cº)

9-14-13

20uL PCR to obtain plasmid backbone using linearized backbone as template.

Gel Legend : 1Kb ladder , 9010 57 C , 9010 57 C(-),  linear 57, linear 57 neg

Week 7

9-15-13

• New PCR of 9010 at 55 Cº and 57 Cº with new GC phusion mastermix due to contamination
• a PCR of Linear PSB1C3 at 57 Cº

gel legend : 9010(55+), 9010(55 C-) , 9010(57 C+), 9010(57 C-), Linear (1), Linear (2), Linear(-), DNA ladder

9-16-13

Gel purification on PCR with linearized backbone

·       Run two samples from the PCR reaction, one with 5uL pcr and loading buffer with Sybr gold, and the other with the rest of the PCR (45uL) and loading buffer without Sybr gold.

·       Load the two samples in side by side lanes

·       Use the Sybr gold in uv light to find the band to cut out of the gel in the next lane over.

·       Centrifuge gel cut out with gel filter to separate. Collect filtrate.

·       Use Zymo research DNA Clean and Concentrate to recover DNA from filtrate

9-17-13

Concentrate DNA

1. Combine all gel purification tubes with purified DNA (add alcohol?)

2. Place separate cap with holes in it on top of tube.

3. Place tube in brick under vacuum chamber.

4. When all liquid is gone, add 5uL of Milli Q water.

9-18-13

Gibson reaction with KR2 G-blocks

         -5uL concentrated plasmid backbone

         -1uL of each G-block

         -3uL H2O

         -10uL 2x Gibson Master Mix

         -PCR Machine 50Cº for 1 hr.

Chemi-competent transformation of E.coli with Gibson reaction products.

• Plate transformed E.coli on LB+Chloramphenicol plates. (400uL, 200uL, 100uL, and one with untransformed E. coli)

9-19-13

Suspend colonies in Milli Q water

• Milli Q water with colonies colony PCR on colonies from 400uL and 200uL plates
• 15 cycles

- 94 Cº initial denature 5 minutes

- 94 Cº denature 15 s

- 55 Cº first anneal 30s

- 68 Cº extension 1:00

• 15 cycles anneal drop down 7 Cº

- 94 Cº denature :15

- 48 Cº second anneal  :30

- 68 Cº extension 1:00

- 68 Cº final extension 5:00

- 4 Cº hold infinite

Inoculation of E. coli colonies in LB + chloramphenicol

9-20-13

Run gel on PCR products from 9-19-13

Gel Legend: 1Kb ladder, “400”, “200”, negative control (no template)

Zymo research plasmid mini-prep from E. coli cultures