(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:

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

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:

• Amount per tube multiplied by 10
  

Pipetting Scheme for Unrestricted reactions:

• 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:

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:

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"