Team:Lethbridge/Notebook/Lab Work/May

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Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

1 May

1.1 May 5/2010

1.2 May 6/2010

1.3 May 10/2010

1.4 May 11/2010 Evening

1.5 May 12/2010

1.6 May 14/2010

1.7 May 17/2010

1.8 May 17/2010 Evening

1.9 May 18/2010

1.10 May 18/2010 Evening

1.11 May 19/2010

1.12 May 20/2010

1.13 May 20/2010 Evening

1.14 May 25/2010

1.15 May 25/2010 Evening

1.16 May 26/2010

1.17 May 27/2010

1.17.1 May 31/2010

1.18 May 31/2010 - Evening

May

May 5/2010

(in the lab: JV)
Objective: Test Restriction Endonucleases for activity (take 2)
Relevant Information:
Plasmid DNA used here will be "ES-pSB-CEYFP" from last year's plasmid stocks
Prefix Enzymes are: EcoRI and XbaI
Suffix Enyzmes are: SpeI and PstI
(JV worked out in lab notebook which buffers would be best for each prefix/suffix enzyme combination)
Reactions will be assembled as follows:

Enzyme Buffer Volume MM(µL) Volume Enzyme(µL)

PstI Red 19.75 0.25

XbaI Tango 19.75 0.25

SpeI Tango 19.75 0.25

EcoRI Red 19.75 0.25

EcoRI/SpeI Red 19.5 0.25 + 0.25

XbaI/SpeI Tango 19.5 0.25 + 0.25

EcoRI/PstI Red 19.5 0.25 + 0.25

XbaI/PstI Tango 19.5 0.25 + 0.25

Make up Master Mixes as follows:

Red MM per tube(µL) Total*(µL)
MilliQ H₂0 15.75 86.675

Red Buffer (10x) 2 11

pDNA** 2 11

Tango MM per tube(µL) Total*(µL)
MilliQ H₂0 15.75 86.675

Tango Buffer (10x) 2 11

pDNA** 2 11

Volume per reaction multiplied by 5.5

Unknown concentration of pDNA

Incubated for 70min at 37^oC (Start-1:05pm; End-2:15pm)
Added 3.3µL of 6x loading dye to each reaction mixture and loaded 10µL onto a 1% agarose gel (in TAE)
Added 1µL of 6x loading dye to 2µL of gene ruler 1kb ladder
Load order as follows:

Lane Sample Volume Loaded (µL)
1 pSB-CEYFP/PstI 10

2 pSB-CEYFP/EcoRI 10

3 pSB-CEYFP/EcoRI/PstI 10

4 pSB-CEYFP/EcoRI/SpeI 10

5 pSB-CEYFP/XbaI/PstI 10

6 pSB-CEYFP/XbaI 10

7 pSB-CEYFP/SpeI 10

8 pSB-CEYFP/XbaI/SpeI 10

9 pSB-CEYFP/Red Master Mix Control 10

10 pSB-CEYFP/Tango Master Mix Control 10

11 pSB-CEYFP/MilliQ H₂0 Control 10

12 Ladder 4

Ran gel at 100V for 1 hour

Results:

This gel shows that SpeI does not cut on its own, and does not cut when combined with other enzymes
Conclusion: Test other source of SpeI to see if it has any activity.

May 6/2010

(in the lab: KG, AS)
Objective: To check if the old SpeI enzyme (exp date: March 2011) will cleave plasmid DNA, since we believe the newer SpeI enzyme (exp date: 2012) does not.
Method:

Red Master Mix per tube (µL) Total Volume*

MilliQ H₂0 Water 15.75 63

Red Buffer (10x) 2 8

pDNA** 2 8

Volume per tube multiplied by 4

Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Red Master Mix are: EcoRI+SpeI (old), EcoRI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Tango Master Mix per tube (µL) Total Volume*

MilliQ H₂0 Water 15.75 94.5

Tango Buffer (10x) 2 12

pDNA** 2 12

Volume per tube multiplied by 6

Used pSB NEYFP pDNA from cell E5 in plasmid box

Enzymes that will use Tango Master Mix are: SpeI (old), SpeI (new), XbaI+SpeI (old), XbaI+SpeI (new)
Add 0.25µL of each enzyme to 19.5µL of master mix

Incubated all reactions at 37^oC for 1h (Start-8:30pm; End-9:30pm)
Will not be able to run on agarose gel tonight, will label them so JV can run them in the morning
Tube Names:
Master Mix 1 Control (Red Buffer)
Master Mix 2 Control (Tango Buffer)
E+S(N); EcoRI + SpeI(N)
E+S(O); EcoRI + SpeI(O)
X+S(N); XbaI + SpeI(N)
X+S(O); XbaI + SpeI(O)
S(N); SpeI(N)
S(O); SpeI(O)

Placed in -20^oC freezer of later analysis by agarose electrophoresis

May 10/2010

(in the lab:JV)
Objective: To analyze the restriction test done by KG and AS on May 6/2010 by agarose electrophoresis

Method:

Lane Sample Quantity Loaded (µL)

1 MM1 Control 10

2 MM2 Control 10

3 EcoRI+SpeI(N) 10

4 EcoRI+SpeI(O) 10

5 SpeI(N) 10

6 SpeI(O) 10

7 XbaI+SpeI(N) 10

8 XbaI+SpeI(O) 10

9 1kb Ladder 5

Run gel for 60min at 100V

Results:

It appears as though both SpeI enzymes are working properly here. We will utilize the newer batch of SpeI (expires 2012) from this point forward.

Objective:Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.(JV,KG,AV)
Method:Make 2L of LB media with agar
2x10g Tryptone
2X2.5g Yeast Extract
2x5g NaCl
2x10g Agar

Continued May 11/2010
(Stock Ampicillin solution is 100mg/mL)
Have 4x500mL of LB with Agar
Add 500µL of stock ampicillin to 500mL of media

May 11/2010 Evening

(in the lab: KG, AV, MC, TF, JV, JS)
Objective: To transform the following plasmids into DH5α E.coli cells.

Construct Name (2009)</td> Construct Location (2009)

Lumazine J4

Lumazine-dT J5,J6

sRBS-Lumazine-dT J7,J8

pBAD-TetR I4

pBAD A5,F10

sRBS D5,E10

pSB-CEYFP E5,D6

pSB-NEYFP F5,C6

C-term Tag C10

N-term Tag D9,D10

pTet E4

EYFP A4

CFP Complete D4

Method: Followed Competent Cell Transformation protocol in Common Protocols section and plated on LB agar supplemented with ampicillin.
Results: The following plasmids were successfully transformed and formed colonies:

Lumazine (J4)

sRBS-Lumazine-dT (J7)

sRBS-Lumazine-dT (J8)

pBAD (A5)

pBAD (F10)

pSB-CEYFP

pSB-NEYFP

N-term tag

EYFP (A4)

CFP Complete (D4)

Conclusion: Need another attempt to transform the following plasmids:

Lumazine-dT (J5,J6)

pBAD-TetR

sRBS (D5,E10)

C-Term tag

pTet

May 12/2010

(in the lab: JV)
Objective: Miniprep of plasmid DNA from transformed cells(JV, AV, HB)
Method:

Inoculate 5mL of LB liquid media (with 100µL/mL Ampicillin) with cells from competent cells plates (picked with sterile toothpick).

Allow cells in liquid culture to grow overnight in 37^oC shaking incubator (300RPM) Purify plasmid DNA from cells by using "Boiling Lysis Plasmid Preparation" protocol in Common Protocols Section.

CHANGE: Step 14, used MilliQ H₂O (with 20ng/µL RNase A) instead of TE buffer.

Plasmids were transferred to the "iGEM 2010 - Working Plasmid DNA" box in the -20^oC freezer in the iGEM lab. Plasmids were placed in the following cells:

Construct Cell in Working Plasmid Box (2010) Original Cell in Old Box

sRBS-Lumazine-dT A1 J7

sRNS-Lumazine-dT A2 J8

CFP Complete B6 D4

Lumazine A3 J4

pBAD A4 A5

pBAD A5 F10

pSB-CEYFP B5

pSB-NEYFP B4

EYFP B1 A4

N-term tag B2

Also generated sterile glycerol stocks and placed in -80^oC freezer in the 2010 iGEM box as follows:

Construct Cell Working Glycerol Stock Box (2010)

sRBS-Lumazine-dT (J7) B2,C4,D2

sRNS-Lumazine-dT (J8) C6

CFP Complete A10, C8

Lumazine A8,B10

pBAD (from A5) B5,B9

pBAD (from F10) B3,B7

pSB-CEYFP C3,B5

pSB-NEYFP B6,C1

EYFP C7,B8

N-term tag C2,D4

Objective: Restrict plasmid DNA with restriction endonucleases (JV)
Method:
Have: 10 lanes of restricted plasmid DNA
10 lanes of unrestricted plasmid DNA
1 lane of buffer control
Use EcoRI (prefix cutter) and PstI (suffix cutter)

Pipetting Scheme for Restriction Tubes:

Ingredient Volume/tube (µL) Total Volume*

MilliQ H₂O 15.5 155

Red Buffer (10X) 2 20

EcoRI 0.25 2.5

PstI 0.25 2.5

Amount per tube multiplied by 10

Pipetting Scheme for Unrestricted reactions:

Ingredient Volume/tube (µL) Total Volume*

MilliQ H₂O 16 160

Red Buffer (10X) 2 20

Amount per tube multiplied by 10

Buffer Control will be 18µL MilliQ H₂O + 2µL 10x Red Buffer.
Place in 37^oC water bath at 2:55pm and removed at 4:57pm for a 2 hour incubation.
Analyzed restriction digests on a 1% agarose gel (large gel apparatus ~70mL)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 2µL of 6x DNA loading dye to 6µL of TAE buffer and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:

Lane</td> Sample</td> Volume Loaded (µL)

1 1 kb Ladder 5

2 Buffer Control 5

3 pSB-NEYFP 5

4 Restricted Lumazine 5

5 Lumazine 5

6 Restricted pSB-NEYFP 5

7 pSB-CEYFP 5

8 Restricted pSB-CEYFP 5

9 pBAD 5

10 Restricted pBAD 5

11 EYFP 5

12 Restricted EYFP 5

13 CFP Complete 5

14 Restricted CFP Complete 5

15 sRBS-Lumazine-dT (J7) 5

16 Restricted sRBS-Lumazine-dT (J7) 5

17 N-term Tag 5

18 Restricted N-term Tag 5

19 sRBS-Lumazine-dT (J8) 5

20 Restricted sRBS-Lumazine-dT (J8) 5

Ran gel at 100V for 90 minutes (Start-9:50pm; End-11:20pm)
Stained with ethidium bromide for 20 minutes.

Results:

May 13/2010 Evening(in lab: AS,TF,KG,JS,MC)
Objective: To make a second attempt at transforming plasmids that didn't transform the first time. These plasmids are:

Lumazine-dT (J5,J6)

pBad-TetR

sRBS (D5,E10)

C-term tag

pTet

All DH5α cells were used up in the last transformation, had to aliquot an additional 50x 20µL aliquots (MC,TF)
Transform plasmid DNA (Using "Competent Cell Transformation" Protocol) into newly aliquotted DH5α cells. (KG,JS)
NOTES:
AS concerned that there is something not quite right with LB liquid media added to transformed cells, but continued anyways (JV informed AS the next day that the LB liquid media had not been sterilized).
Plated all 250µL of culture.
Results:

Construct</td> Result

Lumazine-dT(1) Growth present

sRBS-Lumazine-dT Growth present

sRBS (D5) Growth present

sRBS (E10) Growth present

C-term tag No growth present

pTet No growth present

Next Steps:
Make another attempt to transform the C-term tag and pTet constructs.
Start overnight cultures of cells that grew for plasmid prep and sequencing.

May 14/2010

(in the lab: JV)
Objective: Quantify pDNA concentration in order to ensure sufficient material for sequence analysis.
Method: Measure absorbance of samples at 260nm.
Results:

Sample</td> Absorbance at 260nm

sRBS-Lumazine-dT (J7) 0.311

sRBS-Lumazine-dT (J8) 0.309

CFP complete 0.316

N-term tag 0.290

pSB-CEYFP 0.338

pSB-NEYFP 0.403

pBAD (A5) 0.282

pBAD (F10) 0.562

EYFP 0.389

Lumazine 0.221

Conclusion: All plasmids present in sufficient concentrations for sequence analysis.

Objective: Purify plasmid DNA from cells recently transformed.
Method:

Inoculate 5mL of sterile LB liquid media (with 100µg/mL ampicillin) with cells picked from colonies of transformation plates, including the following:
Lumazine-dT (J5)
pBad-TetR
sRBS (D5,E10)

NOTE: Lumazine-dT did NOT grow overnight

Followed "Boiling Lysis Plasmid Preparation (Miniprep)" protocol. (May 15/2010; JV,TF)
NOTE: Added 50µL of MilliQ H₂O (with RNase A at a concentration of 20ng/µL) to dissolve pDNA instead of TE buffer.

Objective: Perform restriction digest on the above prepared plasmid DNA.
Method:
Used EcoRI as prefix cutter and PstI as suffix cutter.
Pipetting Scheme for Restriction Tubes:

Ingredient Volume/tube (µL) Total Volume*

MilliQ H₂O 16 56

Red Buffer (10X) 2 7

EcoRI 0.25 0.875

PstI 0.25 0.875

Amount per tube multiplied by 3.5

Add 18µL master mix to each plasmid DNA sample
Pipetting Scheme for Unrestricted reactions:

Ingredient Volume/tube (µL) Total Volume*

MilliQ H₂O 16 56

Red Buffer (10X) 2 7

Amount per tube multiplied by 3.5

Add 18µL master mix to each plasmid DNA sample
Buffer Control will be 18µL MilliQ H₂O + 2µL 10x Red Buffer.
Place in 37^oC water bath at 12:37pm and removed at 1:55pm for approximately 1 hour incubation.
Analyze samples on a 1% agarose gel (small gel apparatus).
Add 3.3µL of 6x DNA loading dye to each reaction mixture and load.

Lane</td> Sample</td> Volume Loaded (µL)</td>

1 1 kb Ladder 4

2 Restricted sRBS (E10) 10

3 sRBS (E10) 10

4 Restricted sRBS (D5) 10

5 sRBS (D5) 10

6 Restricted sRBS-Lumazine-dT 10

7 sRBS-Lumazine-dT 10

8 Red Buffer Control 10

Ran gel at 100V for 75 minutes (Start-2:30pm; End-3:45pm)
Stained in ethidium bromide for 10 minutes

Results:

There is plasmid DNA in each sample which, when cut with both the prefix and suffix enzyme, yields a band approximately 2000bp (size of pSB1A3 is 2157bp).

May 17/2010

(in the lab: JV, AV)
Make agar plates with 100µg/mL of ampicillin
Make 5 x 5mL sterile liquid SOC broth
Make 13 x 5mL sterile liquid LB broth

May 17/2010 Evening

(in the lab: TF, AS)
Objective: To grow cells for future use
Streaked plates from glycerol stocks of the following:
iGEM 2007 -80^oC Freezer Box:

Construct Cell type Location of cells

xylE DH5α C4

xylE BL21(DE3) B4

C-term Bba DH5α H4

C-term Bba DH5α I4

C-term Bba DH5α J4

Mr. Gene mms6 DH5α A6

Mr. Gene mms6 DH5α B6

iGEM 2010 -80^oC Freezer Box:

Construct Cell type Location of cells

pLacI DH5α B1

dT BL21(DE3) D1

Incubated at 37^oC, beginning at 20h00 (8:00pm)

Made liquid cultures from cells taken from transformation plates (grown on May 13/2010):

sRBS (D5)

sRBS(E10)

Lumazine-dT (1)

pBad-TetR

Incubated at 37^oC, beginning at 20h30 (8:30pm); shaking at 300RPM
Results:
All streak plates (with cells from glycerol stocks) grew.
Only sRBS (D5) and pBad-TetR cells (from transformation plates) grew.

May 18/2010

(in the lab: JV, AV, HB)
NOTE: Cells from liquid cultures grown last night (May 17/2010) were made into glycerol stocks and placed into the working glycerol stock box as follows:

pBAD-TetR - E5

pBAD-TetR - E6

sRBS (D5) - E7

sRBS (D5) - E8

Objective: To isolate plasmid DNA of pBad-TetR and sRBS (D5) and cut with restriction enzymes.

Method:
Use boiling lysis miniprep to prepare plasmid DNA. Digest sRBS with PstI only; digest pBAD-TetR with SpeI (old and new) and PstI.

Reaction conditions for PstI using Orange Buffer.

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.75

Orange Buffer 2

Plasmid DNA 2

PstI 0.25

Reaction conditions for PstI using Tango Buffer.

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.75

Tango Buffer 2

Plasmid DNA 2

PstI 0.25 & 0.25

Objective: To Restrict pLacI (D2) and sRBS-Lumazine Synthaze-dT (A2) and ligate them together

Method:
Reaction conditions for the Plasmid DNA control.

Ingredient Volume/tube (µL)

Milli-Q H₂O 16

Tango Buffer 2

Plasmid DNA (pLacI or sRBS-Lum-dT) 2

Reaction conditions for XbalI, and PstI using Tango Buffer.

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Tango Buffer 2

Plasmid DNA (sRBS-Lum-dT) 2

XbaI & PstI 0.25 & 0.25

Reaction conditions for SpeI, and PstI using Tango Buffer.

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Tango Buffer 2

Plasmid DNA (sRBS-Lum-dT) 2

SpeI & PstI 0.25 & 0.25

Buffer control contains: 18µL Milli-Q H₂O, and 2µL Tango Buffer.

Incubated at 37^oC from 1:30pm to 2:55pm.
Analyze results on 1% Agarose gel (in 1x TAE); NOTE: Sample mixed with loading dye prior to loading onto gel.

Lane Sample Volume Sample (µL) Volume Dye (µL)

1 pBad-TetR Restricted with old SpeI 5.0 1.0

2 pBad-TetR Restricted with new SpeI 5.0 1.0

3 pBad-TetR Unestricted (Tango Buffer) 5.0 1.0

4 pBad-TetR Restricted with PstI 5.0 1.0

5 pBad-TetR Unrestricted (Orange Buffer) 5.0 1.0

6 sRBS Restricted with PstI 5.0 1.0

7 sRBS Unrestricted 5.0 1.0

8 Buffer Control (Tango) 5.0 1.0

9 Buffer Control (Tango) 5.0 1.0

10 Empty 5.0 1.0

11 Empty 5.0 1.0

12 Empty 5.0 1.0

13 Empty 5.0 1.0

14 Empty 5.0 1.0

15 Empty 5.0 1.0

16 sRBS-Lum-dT Unrestricted 5.0 1.0

17 sRBS-Lum-dT Restricted with XbaI/PstI 5.0 1.0

18 pLacI Unrestricted 5.0 1.0

19 pLacI Restricted with SpeI/PstI 5.0 1.0

20 1kb Ladder 2.5 0.5

Ran gel at 100V for 90 minutes.
Results:

Objective: Make liquid cultures of streak plates (made May 17/2010) for plasmid mini-preps.
Method: Added 5µL of 100mg/mL ampicillin to 5mL of LB liquid broth to give a final concentration of 100µg/mL ampicillin.
Picked cells from single colonies and inoculated into 5mL LB (Amp⁺) media of the following constructs:

C-term BBa (I4-2007 Box)

C-term BBa (J4-2007 Box)

C-term BBa (H4-2007 Box)

dT (D1-2010 Box)

pLacI (B1-2010 Box)

mr. Gene mms6 (A6-2007 Box)

mr. Gene mms6 (B6-2007 Box)

xylE (B4-2007 Box)

xylE (C4-2007 Box)

May 18/2010 Evening

(in the lab: KG)
Objective: Restrict pLacI, sRNS, sRBS-Lumazine Synthase-dt out of plasmid then ligase pLacI and sRBS also pLacI sRBS-Lumazine Synthase-dt.

Method:
Restriction Digestion
Tube 1 contains:

Ingredient Volume/tube (µL)

Milli-Q H₂O 31.50

Red Buffer 4

Plasmid DNA (pLacI) 4

EcoRI & SpeI 0.50 & 0.50

Tube 2 contains:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.75

Tango Buffer 2

Plasmid DNA (sRBS) 2

XbaI & PstI 0.25 & 0.25

Tube 3 contains:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.75

Red Buffer 2

Plasmid DNA (pLacI) 2

XbaI & PstI 0.25 & 0.25

Incubated at 37^oC for 1 hour starting at 7:00pm.
After incubation tubes 1,2, and 3 were placed on a heating bloack for 10 minutes at 65^oC (Start-8:08pm and 8:18pm). Also heated samples from JV restriction with pLacI only cut at the suffix.

Ligation
Tubes 4,5, and 6

Ingredient Volume/tube (µL)

Ligation Buffer 2

Milli-Q H₂O 17

T4 DNA Ligase 1

Tube 4 contains:

sRBS from Restriction</td><td></table>
Tube 5 contains:

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

PlacI from restriction(double cut) 7.5

sRBS-Lumazine Synthase-dt

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

PlacI from restriction(double cut) 7.5

Tube 6 contains:

sRBS-Lumazine Synthase-dt

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

PlacI from restriction(single cut) 7.5

Begin room temperature incubation at 8:25pm.

May 19/2010

(in the lab:JV)

Objective:Isolate and restrict plasmid DNA from liquid cultures started May 18, 2010.

Method:
Use boiling lysis miniprep to prepare plasmid DNA.

C-term BBa (I4-2007 Box)

C-term BBa (J4-2007 Box)

C-term BBa (H4-2007 Box)

dT (D1-2010 Box)

pLacI (B1-2010 Box)

mr. Gene mms6 (A6-2007 Box)

mr. Gene mms6 (B6-2007 Box)

xylE (B4-2007 Box)

xylE (C4-2007 Box)

Restriction Digestion of prepared plasmid DNA
Master Mix 1:

Ingredient Volume/tube (µL) (Volume/tube(µL)) X 10

Milli-Q H₂O 15.75 157.5

Orange Buffer 2 20

EcoRI 0.25 2.5

Master Mix 2:

Ingredient Volume/tube (µL) (Volume/tube(µL)) X 10

Milli-Q H₂O 16 160

Orange Buffer 2 20

Add 2(µL) of plasmid DNA to 18(µL) of master mix to create restriction digestion reaction.
Buffer control contains 2(µL) of orange buffer and 18(µL) Milli-Q H₂O.
Reactions ran for 1 hour at 37^oC.

May 20/2010

(in the lab:JV, AV)

Objective:Check plasmid DNA and restriction digest reactions prepared May 19,2010.

Method:
Analyzed restriction digests on a 1% agarose gel (large gel apparatus)
Added 1µL of 6x DNA loading dye to 5µL of sample
Added 1µL of 6x DNA loading dye to 4µL of Milli-Q H₂O and 1µL of 1kb DNA mass ladder.
Loaded samples as follows:

Lane Sample Volume Loaded (µL)

1 1 kb Ladder 5

2 Buffer Control 5

3 pLacI (B1) restricted 5

4 pLacI (B1) unrestricted 5

5 dT (B1) restricted 5

6 dT (B1) unrestricted 5

7 mms6 (A6) restricted 5

8 mms6 (A6) unrestricted 5

9 mms6 (B6) restricted 5

10 mms6 (B6) unrestricted 5

11 xylE (B4) restricted 5

12 xylE (B4) unrestricted 5

13 xylE (C4) restricted 5

14 xylE (C4) unrestricted 5

15 C-term (H4) restricted 5

16 C-term (H4) unrestricted 5

17 C-term (J4) restricted 5

18 C-term (J4) unrestricted 5

19 C-term (I4) restricted 5

20 C-term (I4) unrestricted 5

Ran gel at 100V for 2 hours
Results:

May 20/2010 Evening

(in the lab:JS, AS)

Objective: Re-run the gel from earlier in the day with a higher DNA concentration

Method:
Analyzed restriction digests on a 1% agarose gel (large gel apparatus)
Added 2µL of 6x DNA loading dye to 10µL of sample
Added 2µL of 6x DNA loading dye to 8µL of Milli-Q H₂O and 2µL of 1kb DNA mass ladder.
Loaded samples as follows:

Lane Sample Volume Loaded (µL)

1 1 kb Ladder 5

2 pLacI (B1) unrestricted 10

3 pLacI (B1) restricted 10

4 dT (B1) unrestricted 10

5 dT (B1) restricted 10

6 mms6 (A6) unrestricted 10

7 mms6 (A6) restricted 10

8 mms6 (B6) unrestricted 10

9 mms6 (B6) restricted 10

10 xylE (B4) unrestricted 10

11 xylE (B4) restricted 10

12 xylE (C4) unrestricted 10

13 xylE (C4) restricted 10

14 C-term (H4) unrestricted 10

15 C-term (H4) restricted 10

16 C-term (I4) unrestricted 10

17 C-term (I4) restricted 10

18 C-term (J4) unrestricted 10

19 C-term (J4) restricted 10

20 Buffer Control 10

Ran gel for 80 minutes at 100V.
Results:

May 25/2010

(in the lab:JV, HB, AV)

Objective: Transform the following ligated parts:

pLacI-sRBS (single cut from May 18,2010)

pLacI-sRBS (double cut from May 18,2010)

pLacI-sRBS-lumazine synthase-dt (from May 18, 2010)

Method:
Used Competent Cell Transformation to prepare plasmid DNA.
Results:
No colonies grew. No colonies on positive control plate. Something amiss here.
NOTE (June 1/2010; AS): Likely that the problem here was the method used to heat shock the cells. Cells were heat shocked in a heat plate, with incomplete contact of the heating surface, causing inefficient transfer of heat to cells for shock and movement of DNA into competent cells.

May 25/2010 Evening

(in the lab: AV, KG)

Objective:Restriction of dt, xylE, and mms6. To ligate xylE with dt and mms6 with dt.

Method:

Cut dt with XbaI and PstI (Buffer: Tango)

Cut xylE with EcoRI and SpeI (Buffer: Red)

Cut mms6 with EcoRI and SpeI (Buffer: Red)

Restriction of above plasmids.
Tube 1:

Ingredient Volume/tube (µL) (Volume/tube(µL)) X 4

Milli-Q H₂O 15.5 155

Tango 2 8

plasmid (dt) 2 8

XbaI & PstI 0.25 & 0.25 0.5 & 0.5

Tube 2:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Red 2

plasmid (mms6(A3)) 2

EcoRI & SpeI 0.25 & 0.25

Tube 3:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Red 2

plasmid (mms6(B3)) 2

EcoRI & SpeI 0.25 & 0.25

Tube 4:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Red 2

plasmid (xylE(B4)) 2

EcoRI & SpeI 0.25 & 0.25

Tube 5:

Ingredient Volume/tube (µL)

Milli-Q H₂O 15.5

Red 2

plasmid (xylE (C4) 2

EcoRI & SpeI 0.25 & 0.25

Incubated on heat block set at 37^oC between 7:19-8:19pm

Objective: Ligate mms6 to dt, and xylE to dt (JV, AV, HB, KG, AS)

Method:
Tube 1:

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

mms6 (B6) 7.5

dt (B1) 7.5

Tube 2:

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

mms6 (A6) 7.5

dt (B1) 7.5

Tube 3:

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

xylE (B4) 7.5

dt (B1) 7.5

Tube 4:

Ingredient Volume/tube (µL)

Milli-Q H₂O 2.75

Ligation Buffer 2

T4 DNA Ligase 0.25

xylE (C4) 7.5

dt (B1) 7.5

T4 DNA ligase added to ligations at 8:37pm.
Incubated reactions overnight at room temperature
Inactived by heating for 10 minutes at 80^oC at 10:00am

Analyze restrictions and ligations by electrophoresis on a 1% agarose gel, stained with ethidium bromide. Load order is as follow:

Lane Sample Volume Rxn (µL) Volume Dye (µL)

1 Restricted mms6 (B6) 10 10

2 Unrestricted mms6 (B6) 10 10

3 Restricted mms6 (A6) 10 10

4 Unrestricted mms6 (A6) 10 10

5 mms6 (B6)-dT ligation 10 10

6 mms6 (A6)-dT Ligation 10 10

7 Restricted dT (B1) 10 10

8 Unrestricted dT (B1) 10 10

9 Restricted xylE (C4) 10 10

10 Unrestricted xylE (C4) 10 10

11 Restricted xylE (B4) 10 10

12 Unrestricted xylE (B4) 10 10

13 xylE (C4)-dT Ligation 10 10

14 xylE (B4)-dT Ligation 10 10

15 1kb Ladder 2 (+8water) 2

16 Empty

17 Empty

18 Empty

19 Empty

20 Empty

Gel was run at 100V for 2 hours
Results:

Objective:Grow overnight cultures of cells for pDNA mini-prep tomorrow.
Method:
DH5α cells from -80^oC freezer containing the below genes were inoculated into 5mL LB liquid broth containing 100µg/mL of ampicillin and subsequently incubated at 37^oC with shaking (at 300RPM) overnight (beginning at 7:30pm)
Cells with the following gene products were retrieved from the -80^oC freezer:

Common Name Box Location Result

Fusion CEYFP 2007 H5,I5,J5 All grew

CEYFP 2007 G6,H6 All grew

NEYFP 2007 I6,J6 All grew

Arg N-term 2007 I7,J7 None grew

Arg C-term 2007 J8 All grew

ECFP 2009 A3,B3,C3 All grew

EYFP 2009 A6,B6,C6 All grew

TetR Inverter 2009 A9,B9 None grew

pBad-TetR 2007/2009 G8/C9 All grew

Removed at 9:30pm

May 26/2010

(In the lab: JV)
Objective: Purify plasmid DNA (via mini-prep) of the cells grown last night.
Method: Performed boiling lysis miniprep as described in protocols section on cells containing the following genes:

Fusion CEYFP (H5 - 2007)
Fusion CEYFP (J5 - 2007)
NEYFP (J6 - 2007)
NEYFP (I6 - 2007)
Fusion CEYFP (I5 - 2007)
CEYFP (G6 - 2007)
pBad-TetR (G8 - 2007)
CEYFP (H6 - 2007)
EYFP (A6 - 2009)
ECFP (A3 - 2009)
pBad-TetR (C9 - 2009)
ECFP (C3 - 2009)
EYFP (B6 - 2009)
EYFP (C6 - 2009)
ECFP (B3 - 2009)

Results: Will restrict and run agarose gels tomorrow.

May 27/2010

(In the lab: JV, AV)

Objective:Restrict and run agarose gels of plasmids 'minipreped' yesterday from the 2007 and 2008 glycerol stock boxes.

Method:
Master Mix for Restriction Reactions:

Ingredient Volume/tube (µL) (Volume/tube(µL)) X 16

Milli-Q H₂O 15.75 252

Orange 2 32

EcoRI 0.25 4

Master Mix for unrestricted DNA:

Ingredient Volume/tube (µL) (Volume/tube(µL)) X 16

Milli-Q H₂O 16 256

Orange 2 32

18µL of restriction master mix was added to 2(µL) of each plasmid.
18µL of unrestricted master mix was added to 2(µL) of each plasmid.
18µL of Milli-Q H₂O was added to 2(µL) of orange buffer as a buffer control.
Reactions were carried out for 1 hour at 37^oC.
Analyzed on a 1% agarose gel.

Gel 1

Lane Sample Volume Rxn (µL) Volume Dye (µL)

1 1kb Ladder^† 2 2

2 Buffer Control 10 10

3 CEYFP (H6) Restricted 10 10

4 CEYFP (H6) Unrestricted 10 10

5 CEYFP (G6) Restricted 10 10

6 CEYFP (G6) Unrestricted 10 10

7 EYFP (C6) Restricted 10 10

8 EYFP (C6) Unrestricted 10 10

9 Fusion CEYFP (I5) Restricted 10 10

10 Fusion CEYFP (I5) Unrestricted 10 10

11 EYFP (C3) Restricted 10 10

12 EYFP (C3) Unrestricted 10 10

13 pBad-TetR (C9) Restricted 10 10

14 pBad-TetR (C9) Unrestricted 10 10

15 ECFP (A3) Restricted 10 10

16 ECFP (A3) Unrestricted 10 10

17 Fusion CEYFP (H5) Restricted 10 10

18 Fusion CEYFP (H5) unrestricted 10 10

19 pBad-TetR (G8) Restricted 10 10

20 pBad-TetR (G8) unrestricted 10 10

† Also added 8µL of water to this mix

Gel 2

Lane Sample Volume Rxn (µL) Volume Dye (µL)

1 1kb Ladder^† 2 2

2 Restricted Fusion CEYFP (J5) 10 10

3 Unrestriced Fusion CEYFP 10 10

4 Restricted ECFP (B3) 10 10

5 Unrestricted ECFP (B3) 10 10

6 Restricted EYFP (B6) 10 10

7 Unrestricted EYFP (B6) 10 10

8 Restricted NEYFP (I6) 10 10

9 Unrestricted NEYFP (I6) 10 10

10 Restricted EYFP (A6) 10 10

11 Unrestricted EYFP (A6) 10 10

12 Restricted EYFP (J6) 10 10

13 Unrestricted EYFP (J6) 10 10

† Also added 8µL of water to this mix

Ran both gels at 100V for 2 hours.
Results:
Gel 1

Gel 2

Conclusion: To be concluded....

Objective: Transform DH5α cells with plasmids containing ligation products from May 25/2010.
Method: Transform, using the competent cell transformation protocol, the ligation products with the most intense banding from each of the following ligation reactions:

mms6 + dT (B6 + A6)

xylE + dT (B4 + C4)

Incubate at 37^oC overnight.
Results:
No colonies grew, even on positive control plate.

May 31/2010

Objective: Transform part BBa_J33204 (Ribosomal Binding Site + xylE gene) into DH5α cells.
Method:
Obtained part BBa_J33204 from 2010 iGEM Spring Distribution Kit Plate 1, well 7P.
Transformed using competent cell transformation protocol, along with negative control (milliQ H₂O water) and positive control (pUC19 plasmid). Plated 100µL and 50µL on separate pre-warmed LB agar plates containing 100µg/mL ampicillin.
Added 250µL of SOC media and incubated for 90 minutes at 37^o.
Results:
Obtained the following number of colonies on each plate:

BBa_J33204 50µL - 13 colonies

BBa_J33204 100µL - 18 colonies

pUC19 - 9 colonies

Other lab activities:

Inoculated 5mL of LB liquid broth (Amp⁺ with cells containing Bba_J33204 picked from colony off transformation plate and incubated at 37^oC with shaking overnight.

AV quantified each pDNA stock in the working plasmids box by measuring the A₂₆₀ of a 1:100 dilution of the pDNA samples in a UV cuvette. Results are posted on the working plasmids page.

May 31/2010 - Evening

Objective: Transform recent ligation reactions that didn't work the first time around.
Method: Use competent cell transformation protocol to transform the following ligation products:

pLacI + sRBS

pLacI + sRBS-Lum-dT (times 2)

mms6 (B6) + dT

mms6 (A6) + dT

xylE (C4) + dT

xylE (B4) + dT

Plated all cells; spun down media so cells were pelleted and removed 200uL of cell-free media. Re-suspended pelleted cells in remaining media, and plated onto ampicillin plates.
Results:
No growth on plates EXCEPT:

Positive control (pUC19) - 9 colonies

mms6 (B6) + dT - 1 colonies

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/May"

Enzyme	Buffer	Volume MM(µL)	Volume Enzyme(µL)
PstI	Red	19.75	0.25
XbaI	Tango	19.75	0.25
SpeI	Tango	19.75	0.25
EcoRI	Red	19.75	0.25
EcoRI/SpeI	Red	19.5	0.25 + 0.25
XbaI/SpeI	Tango	19.5	0.25 + 0.25
EcoRI/PstI	Red	19.5	0.25 + 0.25
XbaI/PstI	Tango	19.5	0.25 + 0.25

Red MM	per tube(µL)	Total*(µL)
MilliQ H₂0	15.75	86.675
Red Buffer (10x)	2	11
pDNA**	2	11

Tango MM	per tube(µL)	Total*(µL)
MilliQ H₂0	15.75	86.675
Tango Buffer (10x)	2	11
pDNA**	2	11

Lane	Sample	Volume Loaded (µL)
1	pSB-CEYFP/PstI	10
2	pSB-CEYFP/EcoRI	10
3	pSB-CEYFP/EcoRI/PstI	10
4	pSB-CEYFP/EcoRI/SpeI	10
5	pSB-CEYFP/XbaI/PstI	10
6	pSB-CEYFP/XbaI	10
7	pSB-CEYFP/SpeI	10
8	pSB-CEYFP/XbaI/SpeI	10
9	pSB-CEYFP/Red Master Mix Control	10
10	pSB-CEYFP/Tango Master Mix Control	10
11	pSB-CEYFP/MilliQ H₂0 Control	10
12	Ladder	4

Red Master Mix	per tube (µL)	Total Volume*
MilliQ H₂0 Water	15.75	63
Red Buffer (10x)	2	8
pDNA**	2	8

Tango Master Mix	per tube (µL)	Total Volume*
MilliQ H₂0 Water	15.75	94.5
Tango Buffer (10x)	2	12
pDNA**	2	12

Lane	Sample	Quantity Loaded (µL)
1	MM1 Control	10
2	MM2 Control	10
3	EcoRI+SpeI(N)	10
4	EcoRI+SpeI(O)	10
5	SpeI(N)	10
6	SpeI(O)	10
7	XbaI+SpeI(N)	10
8	XbaI+SpeI(O)	10
9	1kb Ladder	5

Construct Name (2009)</td>	Construct Location (2009)
Lumazine	J4
Lumazine-dT	J5,J6
sRBS-Lumazine-dT	J7,J8
pBAD-TetR	I4
pBAD	A5,F10
sRBS	D5,E10
pSB-CEYFP	E5,D6
pSB-NEYFP	F5,C6
C-term Tag	C10
N-term Tag	D9,D10
pTet	E4
EYFP	A4
CFP Complete	D4

Construct	Cell in Working Plasmid Box (2010)	Original Cell in Old Box
sRBS-Lumazine-dT	A1	J7
sRNS-Lumazine-dT	A2	J8
CFP Complete	B6	D4
Lumazine	A3	J4
pBAD	A4	A5
pBAD	A5	F10
pSB-CEYFP	B5
pSB-NEYFP	B4
EYFP	B1	A4
N-term tag	B2

Construct	Cell Working Glycerol Stock Box (2010)
sRBS-Lumazine-dT (J7)	B2,C4,D2
sRNS-Lumazine-dT (J8)	C6
CFP Complete	A10, C8
Lumazine	A8,B10
pBAD (from A5)	B5,B9
pBAD (from F10)	B3,B7
pSB-CEYFP	C3,B5
pSB-NEYFP	B6,C1
EYFP	C7,B8
N-term tag	C2,D4

Ingredient	Volume/tube (µL)	Total Volume*
MilliQ H₂O	15.5	155
Red Buffer (10X)	2	20
EcoRI	0.25	2.5
PstI	0.25	2.5

Ingredient	Volume/tube (µL)	Total Volume*
MilliQ H₂O	16	160
Red Buffer (10X)	2	20

Lane</td>	Sample</td>	Volume Loaded (µL)
1	1 kb Ladder	5
2	Buffer Control	5
3	pSB-NEYFP	5
4	Restricted Lumazine	5
5	Lumazine	5
6	Restricted pSB-NEYFP	5
7	pSB-CEYFP	5
8	Restricted pSB-CEYFP	5
9	pBAD	5
10	Restricted pBAD	5
11	EYFP	5
12	Restricted EYFP	5
13	CFP Complete	5
14	Restricted CFP Complete	5
15	sRBS-Lumazine-dT (J7)	5
16	Restricted sRBS-Lumazine-dT (J7)	5
17	N-term Tag	5
18	Restricted N-term Tag	5
19	sRBS-Lumazine-dT (J8)	5
20	Restricted sRBS-Lumazine-dT (J8)	5

Construct</td>	Result
Lumazine-dT(1)	Growth present
sRBS-Lumazine-dT	Growth present
sRBS (D5)	Growth present
sRBS (E10)	Growth present
C-term tag	No growth present
pTet	No growth present

Sample</td>	Absorbance at 260nm
sRBS-Lumazine-dT (J7)	0.311
sRBS-Lumazine-dT (J8)	0.309
CFP complete	0.316
N-term tag	0.290
pSB-CEYFP	0.338
pSB-NEYFP	0.403
pBAD (A5)	0.282
pBAD (F10)	0.562
EYFP	0.389
Lumazine	0.221

Lane</td>	Sample</td>	Volume Loaded (µL)</td>
1	1 kb Ladder	4
2	Restricted sRBS (E10)	10
3	sRBS (E10)	10
4	Restricted sRBS (D5)	10
5	sRBS (D5)	10
6	Restricted sRBS-Lumazine-dT	10
7	sRBS-Lumazine-dT	10
8	Red Buffer Control	10

Construct	Cell type	Location of cells
xylE	DH5α	C4
xylE	BL21(DE3)	B4
C-term Bba	DH5α	H4
C-term Bba	DH5α	I4
C-term Bba	DH5α	J4
Mr. Gene mms6	DH5α	A6
Mr. Gene mms6	DH5α	B6

Ingredient	Volume/tube (µL)
Milli-Q H₂O	15.75
Orange Buffer	2
Plasmid DNA	2
PstI	0.25

Lane	Sample	Volume Sample (µL)	Volume Dye (µL)
1	pBad-TetR Restricted with old SpeI	5.0	1.0
2	pBad-TetR Restricted with new SpeI	5.0	1.0
3	pBad-TetR Unestricted (Tango Buffer)	5.0	1.0
4	pBad-TetR Restricted with PstI	5.0	1.0
5	pBad-TetR Unrestricted (Orange Buffer)	5.0	1.0
6	sRBS Restricted with PstI	5.0	1.0
7	sRBS Unrestricted	5.0	1.0
8	Buffer Control (Tango)	5.0	1.0
9	Buffer Control (Tango)	5.0	1.0
10	Empty	5.0	1.0
11	Empty	5.0	1.0
12	Empty	5.0	1.0
13	Empty	5.0	1.0
14	Empty	5.0	1.0
15	Empty	5.0	1.0
16	sRBS-Lum-dT Unrestricted	5.0	1.0
17	sRBS-Lum-dT Restricted with XbaI/PstI	5.0	1.0
18	pLacI Unrestricted	5.0	1.0
19	pLacI Restricted with SpeI/PstI	5.0	1.0
20	1kb Ladder	2.5	0.5

Ingredient	Volume/tube (µL)
Ligation Buffer	2
Milli-Q H₂O	17
T4 DNA Ligase	1

Ingredient	Volume/tube (µL)	(Volume/tube(µL)) X 10
Milli-Q H₂O	15.75	157.5
Orange Buffer	2	20
EcoRI	0.25	2.5

Ingredient	Volume/tube (µL)	(Volume/tube(µL)) X 4
Milli-Q H₂O	15.5	155
Tango	2	8
plasmid (dt)	2	8
XbaI & PstI	0.25 & 0.25	0.5 & 0.5

Lane	Sample	Volume Rxn (µL)	Volume Dye (µL)
1	Restricted mms6 (B6)	10	10
2	Unrestricted mms6 (B6)	10	10
3	Restricted mms6 (A6)	10	10
4	Unrestricted mms6 (A6)	10	10
5	mms6 (B6)-dT ligation	10	10
6	mms6 (A6)-dT Ligation	10	10
7	Restricted dT (B1)	10	10
8	Unrestricted dT (B1)	10	10
9	Restricted xylE (C4)	10	10
10	Unrestricted xylE (C4)	10	10
11	Restricted xylE (B4)	10	10
12	Unrestricted xylE (B4)	10	10
13	xylE (C4)-dT Ligation	10	10
14	xylE (B4)-dT Ligation	10	10
15	1kb Ladder	2 (+8water)	2
16	Empty
17	Empty
18	Empty
19	Empty
20	Empty

Common Name	Box	Location	Result
Fusion CEYFP	2007	H5,I5,J5	All grew
CEYFP	2007	G6,H6	All grew
NEYFP	2007	I6,J6	All grew
Arg N-term	2007	I7,J7	None grew
Arg C-term	2007	J8	All grew
ECFP	2009	A3,B3,C3	All grew
EYFP	2009	A6,B6,C6	All grew
TetR Inverter	2009	A9,B9	None grew
pBad-TetR	2007/2009	G8/C9	All grew

Ingredient	Volume/tube (µL)	(Volume/tube(µL)) X 16
Milli-Q H₂O	15.75	252
Orange	2	32
EcoRI	0.25	4