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April
May

April 13/2010 (In the Lab: JV, AS)
Objective: Test Restriction Endonucleases for Activity
Relevant Information:
Endonucleases available

Endonuclease	Optimal Buffer**	Other Buffers
EcoRV	None	2xT(100%); O,G(50-100%)
EcoRI	Red	O(100%);R(100%)*;2xT(100%)
BcuI/SpeI	Tango	B(50-100%);G(50-100%)
XbaI	Tango	B,G,2xT(50-100%)
PstI	Orange	R(100%); B,G,T,2xT(50-100%)
DpnI	Tango	B,G(100%): O,R,2xT(50-100%)

*Star Activity
**Optimal Buffer from Fermentas

Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme>

Methods: Set up Master Mixes:

Red MM	per tube (µL)	Total (µL)
MilliQ H20	13.75	55
Red Buffer (10x)	2	7
pUC19 (10pg/µL)	2	7
Total	19.75	69

Tango MM	per tube (µL)	Total (µL)
MilliQ H20	13.75	55
Tango Buffer (10x)	2	7
pUC19 (10pg/µL)	2	7
Total	19.75	69

To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37^oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:

Lane	Sample
1	1kb Ladder
2	Tango Control
3	DpnI (Tango)
4	BcuI/SpeI (Tango)
5	XbaI (Tango)
6	EcoRI (Red)
7	PstI (Red)
8	Red Control
9	Empty
10	Empty

Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining


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	.25
XbaI	Tango	19.75	.25
SpeI	Tango	19.75	.25
EcoRI	Red	19.75	.25
EcoRI/SpeI	Red	19.5	.25+.25
XbaI/SpeI	Tango	19.5	.25+.25
EcoRI/PstI	Red	19.5	.25+.25
XbaI/PstI	Tango	19.5	.25+.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.

May 10/2010(in the lab:JV, KG, AV)
Objective:Make 24 LB agar plates with 100µg/mL ampicillin antibiotic.
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)	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	Sample	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	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	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.

Common Protocols:

1. Competent Cell Transformation
2. Boiling Lysis Plasmid Preparation (Miniprep)
   

Competent Cell Transformation

1. Thaw 20µL of aliquotted cells (DH5α of BL21(DE3)) on ice.
2. Gently pipet 2.0µL of DNA into competent cells
   ATTENTION:
   Do not perform any additional mixing
   Never use more DNA that 10% of the volume of the competent cells otherwise the cells get destroyed by osmotic shock
3. Incubate the cells on ice for 30 minutes.
4. Heat shock the cells in a water bath at 42^oC for EXACTLY 45 seconds.
5. Incubate the cells on ice for 1 minute.
6. Add 250µL sterile media to the cells and incubate at 37^oC for 1 hour with shaking (200RPM).
7. Plate 100µL and 50µL on prewarmed LB agar plate containing the appropriate antibiotic.
   For ligations, plate all 250µL.
8. Leave plate for 10-15 minutes to soak the cell suspension into the agar.
9. Flip plate over (agar on top)
10. Incubate the plates in the 37^oC incubator overnight

Boiling Lysis Plasmid Preparation (Miniprep)

1. Aseptically transfer 1.5mL of each overnight culture to a 1.5mL microcentrifuge tube (MCT) and pellet the cells by centrifugation in a benchtop microcentrifuge (2min at 13000RPM)
2. Remove and discard as much of the supernatant as possible by aspiration (e.g with a Pasteur Pipette). Do not suck up the cell pellet!!
3. Rinse the cell pellet by washing 1.0mL of sterile MilliQ H₂O gently down the inside wall of the MCT. This removes any traces of the supernatant adhering to the MCT wall while minimizing the disturbance to the cell pellet. (/li>
4. Resuspend the cell pellet in 350µL of STET.
5. Add 25µL of the Lysozyme solution and mix by inversion.
6. Place the MCT in the bioling water bath for EXACTLY 35 seconds, remove and incubate on ice for 5 minutes.
7. Pellet the cellular debris by centrifugation at 13000RPM for 15 minutes. Transfer the supernatant to a fresh MCT and discard the pellet.
8. Precipitate the plasmid DNA by adding 40µL of 3.0M sodium acetate (pH 5.2) and 420µL isopropanol. Mix by inversion. Mix by inversion and incubate for 5 minutes at room temperature.
9. Pellet the plasmid DNA by centrifugation at 13000RPM for 10 minutes at 4^oC. A pellet of plasmid DNA should be visible at the base of the MCT when complete.
10. Being careful not to disturb the pellet, discard the supernatant and rinse the pellet with 500µL of ice cold ethanol.
11. Repeat above step.
12. Invert and tap the open MCT several times against a piece of paper towel on your bench to remove as much ethanol as possible.
13. Store the open MCT at room temperature for approximately 10 minutes to allow all remaining traces of ethanol to evaporate
14. Add 50µL of TE (pH 8.0) containing RNase A and resuspend the plasmid DNA by flicking the base of the MCT with your finger. The plasmid DNA is ready for use or can be stored long term at -20^oC.