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From 2014.igem.org

George's Lab Notebook

6/9/14

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PCR for Constitutive Promoters (m10)

Materials	1x reaction	4.5x Master Mix
x Phusion HF Buffer	10 µl	45 µl
dNTP's (10 mM)	1 µl	4.5 µl
Forward Primer (10µm)	2.5 µl	11.25 µl
Reverse Primer (10µm)	2.5 µl	11.25 µl
*Template DNA (m10)	0.3 µl	1.35 µl
Phusion Polymerase	0.5 µl	2.25 µl
Water	33.2 µl	149.4 µl

Total	50 µl	225 µl

1. Mix materials in a 4.5x Master Mix on ice. Mix well.
2. Pipetter 50 µl from the Master Mix into 4 labeled PCR tubes
3. Thermocycler for : 

    Initial Duration   | 98° C | 30s 
      35 Cycles of: 
          Denaturation | 98° C | 10s
          Annealing    | 55° C | 20s
          Extension    | 72° C | 30s
    Final Extension    | 72° C | 5m
    Hold               | 4°  C | Forever

Possible Errors:
Incomplete thawing of dNTPs, not well mixed

Working Stock of Reverse and Forward Primers for pTEF1 kept in my freezer box. 6/9/14

6/10/14

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Gel loading of pTEF1 in Gel

Loading order: 15Wells 100bp ladder|Eleanorx4|Robertx4|Sabrinax4|100bp ladder

20wells 100bp ladder|Jeffreyx5|skip|Georgex2|100bp ladder

*m10 only had two wells loaded because a centrifuge spin burst onpen my caps and 2 flew out. Luckily one was unharmed and I was able to load two samples in.

Gel Extraction

Cut DNA band from PCR of constitutive promoter gel
Weigh gel: .280g
QG Buffer: 840ul
    *QG buffer amount is 3:1 of gel weight

1. Mix gel slice with QG Buffer in 50C heat bath for 10 min.
2. Add 1 ml isopropanol. Mix.
3. Add 750 ul of mixture into purple Qiagen spin column. Spin for 30 sec. Discard liquid.  Repeat.
4. Add 750 ul of Buffer PE and spin for 1 min. Discard waste.
5. Dry spin the column for 1 min. Replace the spin column in a new microcentrifuge tube
6. Elute in 50 of ddH2O. Wait 1 min and then spin for 1 min.
7.Keep the runthrough and discard the column

Restriction Digest with ApaI

40 ul of DNA (m10 gel extraction)
5 ul of 10x CutSmart Buffer
.5 ul of ApaI Enzyme

*digest overnight at room temperature*

6/11/14

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Restriction Digest with XhoI

Add .5 ul of XhoI enzyme to ApaI digestion from 6/10/14
Incubate in the 37°C shaker.

PCR Purification

1. Add 250 ul of Buffer PB to digestion.
2. Place sample in a purple QIAquick spin column. Spin for 1 min. Discard waste.
3. Wash with 750 ul of Buffer PE. Spin for 1 min. Discard waste.
4. Dry spin for 1 min. Replace spin column in a new microcentrifuge tube.
5. 6. Elute in 50 ul of ddH2O (add directly into the column film). Wait 1 min and then spin for 1 min.

Total concentration: 40.28ul

DNA Ligation

Materials	Volume
10x Ligase Buffer	1 ul
DNA Backbone PSV606	.2 ul
DNA Insert (PCR Purication)	.2 ul
T4 DNA Ligase	.5 ul
H2O	8.1 ul

Total                       | 10 ul
----------------------------------------------------
Mix reagents and incubate at room temperature for 2 hrs

Ligation of pTEF1 into PSV606 kept in my freezer box. 6/11/14

Transformation

10 ul of ligation
50 ul of E. Coli competent cells

30 min    | ice
45 sec    | 42°C heat shock
2 min     | ice

Add 250 ul of SOC media. Incubate at 37°C for 1 hr.
Plate on LB+Carb. 

Only 2 colonies formed on plate 

6/12/14

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Redo Transformation

Follow procedure from 6/11/14 with minor alterations.
1. Use .4 ul of ligation instead of .2 ul.
2. Use more competant cells.
    *REMEMBER NEGATIVE CONTROL GUYS

6/13/14

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Transforming α-inducible promoters

Transforming 11 different inducible promoters into CB008 and CB008DB strains of yeast.

1. Boil salmon sperm DNA (ssDNA) for 10 min.-->10 ul of 10 mg/ml stock per transformation
2. Cool ssdna on ice for at least 10 minutes
3. Pellet yeast cultures in centrifuge. (3000 rpm for 2-5 min.)
4. Resuspend with 1ml .1M LiOAc in TE.
5. Pellet cells (3000 rpm for 2-5 min.)
6. Resuspend in 100 ul .1M LiOAc in TE per 2.5 ml culture. 
7. Aliquot 100 ul into each microcentrifuge per transformation. (22 tubes)
Per Tube:
8. Add 100 ug ssDNA, 1 ug of target DNA (1~5 ul)

ADD IN ORDER
9. 480 ul 50% PEG 3350
10. 60 ul 10x TE
11. 60 ul 1 M LiOAc
12. 75 ul DMSO

13. Vortex
14. Incubate at 42°C for 30 min. 
15. Pellet cells (6000 rpm for 2 min)
16. Resuspend with 500 ul YPD.
17. Pellet cells.
18. Resuspend with 50 ul  YPD
19. Plate on SDS-Ura. 

Incubate 2 days at 30°C.

6/16/14

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Colony PCR for Screening E. coli

1. Pick a single colony using a sterile wooden stick and mix in 25ul of water in a PCR tube. Do this for about 4-6 colonies. Use 5ul for the PCR reaction below, adn save the rest for later.

2. Set up PCR reaction as below:
                                            1x Reaction      6x master mix
                                        ----------------------------------
2X Go Taq Green PCR Master Mix                  10ul               60ul
10 uM FW primer                                  1ul                6ul
10uM RV primer                                   1ul                6ul
Water                                            3ul               18ul 
Bacterial cells (template)                       5ul                --      


Cycles:
            95                      5 min
            30X:    95              45 s
                    55              30 s
                    72              1 min per kb (adjust accordingly)
            72                      10 min

3. Analyze products on a 1% agarose gel. The GoTaq mix alread has gel loading dye in it, so you can just directly load 5ul of your PCR readction into a gel.

4. For all positive bands on the gel, take the rest of the bacterial cells from step 1 and inoculate them into and overnight LB (+antibiotic) for miniprep the next day.

E. coli Colony PCR results

*refer to gel photo on 6/13/14 in George's Lab notebook-> Gel Photos -> 6-13-14 E. Coli Colony PCR

**Additional Notes: I was m10 so I occupied lanes 11-15 on row 2 and all the results looked quite bomb.

6/16/14

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Sequencing

All sucessful sequences (all of them)

Colony PCR for Yeast + Patching

1. Label the patch plate with the strain and promoter along with the colony number picked. 
2. Pick 3 big isolated colonies and patch with a stick to the patch plate.
3. # the colonies that you picked on the plate.
4. Fill PCR tubes with 10ul of NaOH
5. Stick the rest of the yeast on the stick into the PCR tubes
6. Boil for 10 mins@ 95
7. PCR TIME
8. Set up reactions as followed:

1x Reaction      7x Master Mix
                                        ---------------------------------
    2X Go Taq Green PCR Master Mix              10ul               70ul
    10 uM FW primer                              1ul                7ul
    10uM RV primer                               1ul                7ul
    Water                                        5ul               35ul 
    ----------------------------------------------------------------------
    Boiled Yeast cells (template)                3ul          

9. Set up Thermocycler for:
    95°C       | 5 min
    30x |95°C   | 45 sec
        |50°C   | 30 sec
        |72°C   | 1 min per kb
    72°C       | 10 min
    4°C            | hold

Running a gel for AFRPs

1st gel 
1st row
20wells

1       2       3       4       5       6       7       8       9       10      11      12      13      14      15      16      17      18      19      20
100bb          SAG1                   ECM18                    PRM3                     HYM1                  ASG7                    CLO7            PRM1                                              
ladder  1       2       3       1       2       3       1       2       3       1       2       3       1       2       3       1       2       3       1


2nd row
15 wells
I give up

• Refer to the gel photo in George's noteboot -> Gel photos->6-16-14 AFRPS stuffs i'll update when i find the picture.

6/18/14

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Colony Dilutions

20x dilutions to all CB008 and CB008DB alpha-promoter yeast.

*Note: CB008DB PRM1-2 sticker was labled on top of CB008 SAG1-3 and could've resulted in a sticker coming off at night.

Glycerol stocks

1:20 Dillution
200ul cells + 9.8ml YPD media.
FREEZE

6/19/14

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Flow Cytometry

1. Start overnight cultures of our strains to be tested om SD complete media instead of YPD (YPD has fluorescence).
2. Dilute overnight cultures to OD 0.05-0.1 in the 96-well shakeplate.  The plates are in a cabinet in the limlab wrapped in shiny paper. USe SD complete media and the total volume of the well should be 1mL. Make a plate map now.
3. Allow cells to enter growth phase by putting wells in plate shakers for 3 hours at 1000rpm@30 degrees.
4. Allow induction with alpha factor. The stock alpha factor is 3mM and we used 0, 1, 10, 100, 1000nM. (Stocks cannot be refrozen, discard after use)
5. Allow cells to enter growth stage by putting on plate for 90-120 minutes, no more.96 
6. Transfer 20ul of each culture to the 96 well flow-cytometry v-bottom plate. Use cycloheximide to arrest cells. Cycloheximide suspends cell division.
7. Run on Flow Cytometer
    1. Check liquid box for sufficient amount of liquid, if too low, scream for help.
    2. Check the second tub for liquid with clear top. It should also have some liquid, don;t shake too harshly
    3. Check the gasoline tank, if kinda full dump out and add some bleach to the bottom of the container.
    4. You can open the iGEM 2014 path and the default settings will be default, but check it anyway to be sure.
    5. For each new experiment, load 2 neg control wells and run those first as a blank.

(Voltage settings: FSC:250, SSC:280, FTC:550

Select wells by double-clicking until it is green and you can just add more. The volume can be modified, but 200ul is suggested (decrease by 50ul from our load)

Flow speed is usually kept at either 0.5ul per second or 1ul/second and can be adjusted relatively quickly. VOlume can be less than our sample volume.

Data is exported by usb, no internet is allowed.)

8. Analyze data (graphs: histogram vs FTC,FSC vs SSC) Flowjo note on page 17 and export to matlab yo.

96 well plates