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April
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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 13

Common Protocols:

1. Competent Cell Transformation

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