Team:Lethbridge/Notebook/Lab Work/June

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June 2010

June 1/2010

JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.

Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:

pLacI-sRBS-Lumazine-dT
pLacI-sRBS-Lumazine-dT
mms6 (A6)
mms6 (B6)
xylE (C4)
xylE (B4)

From the 2010 Parts Distribution:

ECFP (Bba_E0020)
EYFP (Bba_E0030)
BglII Endonuclease (Bba_K112106)

June 2/2010

(In Lab: JV)

Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.

Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).

Restriction Reaction

Ingredient	Volume(µL)
MilliQ H₂0 Water	15.75
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2
EcoRI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H₂0 Water	16
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2

DNA was restricted for 80 minutes at 37^oC.

Analyzed results on a 1% agarose gel. Load order as follows:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	Restricted RBS-xylE	10	2
1	Unestricted RBS-xylE^†	1	2
1	1kb Ladder^†^†	2	2

† Added 9µL MilliQ H₂O
†† Added 8µL MilliQ H₂O
Ran gel at 100V from 2 hours.
Results:

Conclusions: Plasmid DNA prep and restriction was successful.

Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:

Restrictions
- Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)
- Restrict the double terminator with XbaI and PstI (Tango Buffer)
- Restrict pSB1T3 with EcoRI and PstI (Red Buffer)
Set up reactions as follows:

Component Volume (µL)

MilliQ H₂O 15.5

Buffer 2

pDNA 2

Enzyme 0.25 + 0.25

Set up control reaction as follows:
- MilliQ H₂O - 16µL
- Buffer - 2µL
- pDNA - 2µL
Incubated reactions for 65 minutes at 37^oC
Killed enzymes by incubating reactions for 10 minutes at 65^oC

Ligation
Reaction set up as follows:

T4 DNA ligase - 0.25µL
rbs-xylE - 5µL
dT - 3µL
pSB1T3 - 8µL
10x Ligation Buffer - 2µL
MilliQ H₂O - 1.75µL

Incubated reactions overnight at room temperature (total of 19.5 hours)
Killed enzymes by incubating reactions for 10 minutes at 80^oC</ul>

June 2/2010 - Evening

Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
Relevant Information:

Want a final mass of 25ng of each pDNA in the ligation mix.
Final concentration of pDNA in restriction digest should be 25-50ng/µL.
Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
Identified the following plasmids in our working plasmids box:

Common Name	Location	Concentration (ng/µL)	Volume/rxn (µL)
pLacI Maxiprep	A9	990	~1
pLacI (B1)	A6	440	~2
sRBS-Lum-dT (2)	A1	965	~1
sRBS-Lum-dT (1)	A2	1145	~1
sRBS-Lum-dT Maxiprep	B8	4780	~.2
sRBS-Lum-dT	B7	4375	~.25
sRBS-Lum-dT (1)	G2	335	~3
sRBS-Lum-dT (2)	G3	965	~2

Make a 1:10 dilution of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5µL pDNA in 4.5µL water.
Cut pLacI with EcoRI and SpeI
Cut sRBS-Lum-dT with XbaI and PstI
Cut pSB1T3 with EcoRI and PstI
Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.

Method:
Restriction

Name	[pDNA] (ng/µL)	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (A2)	1145	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
pLacI Maxiprep (A1)	990	1	43.5	5	0.25µL EcoRI 0.25µL SpeI	50
sRBS-Lum-dT Maxiprep(B8)	4780	2 (of 1:10 dilution)	42.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (B7)	4375	2.5 (of 1:10 dilution)	42	5	0.25µL XbaI 0.25µL PstI	50
pLacI (D6)	440	2	42.5	5	0.25µL EcoRI 0.25µL SpeI	50
sRBS-Lum-dT (G2)	335	3	41.5	5	0.25µL XbaI 0.25µL PstI	50
sRBS-Lum-dT (G3)	540	2	42.5	5	0.25µL XbaI 0.25µL PstI	50
pSB1T3	25	12.5	7	5	0.25µL EcoRI 0.25µL PstI	50

Incubate for 30 minutes at 37^oC (Start- 12:10pm; End- 12:40pm)
Heat kill enzymes at 80^oC for 20 minutes

Ligation:
In a 10µL final volume, add:

2µL of sRBS-Lum-dT component
2µL of pLacI component
2µL of pSB1T3 component
1µL of T4 Buffer
0.25µL of T4 DNA Ligase
2.75µL of MilliQ H₂O

Incubate for 30 minutes at room temperature to ligate
Incubate for 20 minutes at 80^oC to heat kill

June 3/2010

Carried out protocol described in June 2/2010 - Evening
Analyzed results on 1% agarose gel.Load order as follows:

Lane	Gel 1 Sample	Gel 1 Load	Gel 2 Sample	Gel 2 Load
1	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H₂O	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H₂O
2	Restricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
3	Unrestricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
4	Restricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
5	Unrestricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
6	Restricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
7	Unrestricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
8	Restricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
9	Unrestricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
10	Restricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
11	Unrestricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
12	Restricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
13	Unrestricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
14	Restricted pLacI (A9)	10µL DNA 2µL Dye	Restricted rbs-xylE	10µL DNA 2µL Dye
15	Unrestricted pLacI (A9)	10µL DNA 2µL Dye	Unrestricted rbs-xylE	10µL DNA 2µL Dye
16	Restricted pLacI B1 (D6)	10µL DNA 2µL Dye	Restricted pSB1T3	10µL DNA 2µL Dye
17	Unrestricted pLacI B1 (D6)	10µL DNA 2µL Dye	Unrestricted pSB1T3	10µL DNA 2µL Dye
18	Unrestricted dT	10µL DNA 2µL Dye	pSB1T3 Ligation of: rbs-xylE+dT	10µL DNA 2µL Dye
19	Restricted dT	10µL DNA 2µL Dye

Ran gel at 100V for 90 minutes.
Results:

Conclusions:

June 3/2010 - Evening

Objective: Repeat restriction of pSB1T3 and ligate with pLacI and sRBS-Lum-dT. Previous ligations all used up on gel.
Method:
Ligation:
In a 10µL final volume, add:

2µL of sRBS-Lum-dT component
2µL of pLacI component
2µL of pSB1T3 component
1µL of T4 Buffer
0.25µL of T4 DNA Ligase
2.75µL of MilliQ H₂O

Incubate for 30 minutes at room temperature to ligate
Incubate for 20 minutes at 80^oC to heat kill
Following ligation, transformed using transformation protocol. Plates incubated in 37^oC incubator for 44 hours.
Results: Only plate pLacI (D6) + sRBS-Lum-dT (G2) + pSB1T3 grew; had 2 colonies. Control plate did not grow, acidentally plated on tetracycline plate instead of ampicillin (pUC19).
Follow-up: Inoculated 5mL LB media (tetracycline positive) with cells from the transformation plates and incubated at 37^oC overnight. (June 5/2010).

Objective: Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.
Method:
Restriction

Name	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
pSB-NEYFP (B4)	.8	43.7	5	0.25µL XbaI 0.25µL PstI	50
pSB-CEYFP (B5)	.9	43.6	5	0.25µL XbaI 0.25µL PstI	50
pBad-TetR)	.3	44.2	5	0.25µL EcoRI 0.25µL SpeI	50
NEYFP (E1)	4.3	40.7	5	0.25µL XbaI 0.25µL PstI	50
NEYFP (E2)	0.3	42.2	5	0.25µL XbaI 0.25µL PstI	50
Fusion CEYFP (E3)	3.9	40.6	5	0.25µL XbaI 0.25µL PstI	50
Fusion CEYFP (E4)	2.0	42.5	5	0.25µL XbaI 0.25µL PstI	50
Fusion CEYFP (E5)	3.0	41.5	5	0.25µL XbaI 0.25µL PstI	50
CEYFP (E6)	0.6	43.9	5	0.25µL XbaI 0.25µL PstI	50
CEYFP (E7)	0.5	44.0	5	0.25µL XbaI 0.25µL PstI	50
pBad-TetR (F4)	2.5	42	5	0.25µL EcoRI 0.25µL SpeI	50
pBad-TetR (F5)	1.7	42.8	5	0.25µL EcoRI 0.25µL SpeI	50
pSB-CEYFP (G4)	2.9	41.6	5	0.25µL XbaI 0.25µL PstI	50
pSB1C3	15.5	46		0.25µL EcoRI 0.25µL PstI	62

Incubated at 37^oC for 75 minutes.

Used Red buffer for the EcoRI/SpeI and EcoRI/PstI digests
Used Tango buffer for the XbaI/PstI digests
Did not heat kill upon removal from incubation, put directly into -20^oC fridge.

Continue Ligation on Saturday (See below).

June 5/2010

(In the lab:AS)
Objective: Ligate restriction products from June 3/2010.
Relevant information:

Have 3 tubes of part 1 (pBad-TetR)
- In ampicillin backbone
Have 10 tubes of part 2 (fluorescent protein - various)
- In ampicillin backbone
Will have 30 combinations
Will use pSB1C3 as plasmid backbone
- Used most of the pSB1T3 and want to save remainder for creating new backbone via PCR.

Method:
*Restriction digests were not heat killed after reactions. Freezing probably killed the restriction enzymes, but I will hea kill them at 80^oC for 20 minutes anyways prior to adding Ligase.

- Cool on ice for 10 minutes before adding ligase.

Master Mix	Volume/tube (µL)	Total Volume (µL)
DNA	6	---
10x Buffer	1	32
T4 DNA Ligase	.25	8
MilliQ H₂O	2.75	88

Add 4µL master mix to each DNA tube.

Follow-up: Ligation reactions will be transformed into DH5α cells

June 6/2010

(In Lab: JV, HS)

Objective:
Isolate the following plasmid DNA from DH5α:

pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 2)
pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 1)

Method:
Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H₂O and RNase A.

Notes:

Placed colony 2 in cell E10 of glycerol stocks and J6 of working plasmid box.
Placed colony 2 in cell F1 of glycerol stocks and J5 of working plasmid box.

Objective:
Transformed the following plasmid DNA into DH5α cells:

pBad-TetR-CEYFP: (F5+E6), (B10+E7), (B10+E6), (F4+E6), (F5+E4), (F4+E7)
pBad-TetR-Fusion CEYFP: (F5+E3), (B10+E4), (F4+E3), (B10+E3), (B10+E5), (F4+E4), (F5+E4), (F5+E5), (F4+E5)
pBad-TetR-pSB CEYFP: (F4+B5), (B10+G4), (F4+G4), (F4+B5), (F5+B5), (F5+G4)
pBad-TetR-NEYFP: (F5+E1), (B10+E1), (F4+E2), (F5+E2), (B10+E2), (F4+E1)
pBad-TetR-pSB NEYFP: (F5+B4), (F4+B4), (B10+B4)

Positive control -> DH5α + pSB1C3
Negative control -> DH%α + Milli-Q H₂O

Method:
Followed Competent Cell Transformation protocol and used chloramphenicol as an antibiotic. We plated all 200µL of DNA onto the plates. The plates were incubated at 37^oC from 4:30pm to 10:00am.

Results:
None of the plates showed any growth.

June 8/2010

(In the lab: JV)
Objective: Follow the overexpression of our pLacI-sRBS-Lum-dT construct.
Method: FILL ME OUT!!!!!!
Results:

Time	OD₆₀₀ F1	OD₆₀₀ E10
0	0.118	0.103
30	0.133	0.111
30	0.145	0.116
90
120	0.160	0.124
150	0.120	0.093
180	0.129	0.100
210	0.145	0.122
240	0.158	0.145
270	0.171	0.178
300	0.194	0.222
330	0.223	0.280
360	0.252	0.364
390	0.296	0.458
420	0.338	0.557^†
450	0.394	0.656
480	0.453	0.675
510	0.530	0.688
540	0.598^†	0.706
600	0.633	0.752
660	0.653
720	0.679
∞	1.278

† Overexpression induced by adding 1mM IPTG.
Following overexpression, 1mL of cells was removed from the culture, spun down at ~13000xg for 20 seconds, excess media removed and rinsed with water.
Suspended cells in 8M urea, mixed with 6x dye and ran on 18% SDS-PAGE gel for 90 minutes at 200V. Gel stained overnight.
Results: IMAGE TO COME!!!!

Objective: Calculate quantity of DNA in pBad-TetR and fluorescent protein mini-preps by staining an agarose gel.
Method: Restrict plasmid DNA (done by AV,HB,TF on June 7/2010) and run on a 1% TAE agarose gel (JV).

Lane	Gel 1 Sample	Gel 1 Load	Gel 2 Sample	Gel 2 Load
1	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H₂O	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H₂O
2	Restricted EYFP (B1)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E3)	10µL DNA 2µL Dye
3	Unrestricted EYFP (B1)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E3)	10µL DNA 2µL Dye
4	Restricted pSB-CEYFP (B5)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E4)	10µL DNA 2µL Dye
5	Unrestricted pSB-CEYFP (B5)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E4)	10µL DNA 2µL Dye
6	Restricted ECFP (F2)	10µL DNA 2µL Dye	Restricted EYFP (E10)	10µL DNA 2µL Dye
7	Unrestricted ECFP (F2)	10µL DNA 2µL Dye	Unrestricted EYFP (E10)	10µL DNA 2µL Dye
8	Restricted pSB-CEYFP (G4)	10µL DNA 2µL Dye	Restricted pSB NEYFP (B4)	10µL DNA 2µL Dye
9	Unrestricted pSB-CEYFP (G4)	10µL DNA 2µL Dye	Unrestricted pSB NEYFP (B4)	10µL DNA 2µL Dye
10	Restricted EYFP (G1)	10µL DNA 2µL Dye	Restricted ECFP (F3)	10µL DNA 2µL Dye
11	Unrestricted EYFP (G1)	10µL DNA 2µL Dye	Unrestricted ECFP (F3)	10µL DNA 2µL Dye
12	Restricted NEYFP (E2)	10µL DNA 2µL Dye	pBad-TetR (F5)	10µL DNA 2µL Dye
13	Unrestricted NEYFP (E2)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E5)	10µL DNA 2µL Dye
14	Restricted pBad-TetR (B10)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E5)	10µL DNA 2µL Dye
15	Unrestricted pBad-TetR (B10)	10µL DNA 2µL Dye	pBad-TetR (F4)	10µL DNA 2µL Dye
16	Restricted CEYFP (E6)	10µL DNA 2µL Dye	Restricted pSB1T3	10µL DNA 2µL Dye
17	Unrestricted CEYFP (E6)	10µL DNA 2µL Dye	Unrestricted pSB1T3	10µL DNA 2µL Dye
18	Restricted NEYFP (E1)	10µL DNA 2µL Dye		10µL DNA 2µL Dye
19	Unrestricted NEYFP (E1)	10µL DNA 2µL Dye
20	Restricted ECFP (F1)	10µL DNA 2µL Dye
21	Unrestricted ECFP (F1)	10µL DNA 2µL Dye
22	Restricted EYFP (E9)	10µL DNA 2µL Dye
23	Unrestricted EYFP (E9)	10µL DNA 2µL Dye
24	Restricted CEYFP (E7)	10µL DNA 2µL Dye
25	Unrestricted CEYFP (E7)	10µL DNA 2µL Dye
26	Restricted EYFP (E8)	10µL DNA 2µL Dye
27	Unrestricted EYFP (E8)	10µL DNA 2µL Dye

Ran gel at 100V for 90 minutes.
Results:

No bands visible except for pSB1T3 lanes, therefore could not quantify anything that had not already been quantified.
Also, purification of DNA done on June 7/2010 seemed to reduce amount of pDNA in sample.

Objective: Restrict mms6 (D9,D10) and dT (C1) so we can ligate the dT onto the mms6 coding region.
Method:
mms6 Restriction

2µL mms6 pDNA
2µL Red buffer
0.25µL EcoRI
0.25µL SpeI
15.5µL MilliQ H₂O

dT Restriction

2µL dT pDNA
2µL Orange buffer
0.25µL EcoRI
0.25µL XbaI
15.5µL MilliQ H₂O

Incubated for 1 hour at 37^oC
Heat shock on heat block (80^oC) for 20 minutes
Ligation

2µL dT pDNA
2µL mms6 pDNA
0.25µL T4 DNA Ligase
1µL 10x buffer
4.75µL MilliQ H₂O

Analyze results on 1% TAE agarose gel

Lane	Sample	Load (µL)
1	1kb ladder	0.5 ladder; 2 dye; 9.5 MilliQ H₂O
2	Unrestricted mms6 (D9)	10 DNA; 2 Dye
3	Restricted mms6 (D9)	10 DNA; 2 Dye
4	Unrestricted mms6 (D10)	10 DNA; 2 Dye
5	Restricted mms6 (D10)	10 DNA; 2 Dye
6	Unrestricted dT (C1)	10 DNA; 2 Dye
7	Restricted dT (C1)	10 DNA; 2 Dye
8	Empty
9	Empty
10	Empty

Results:

Looks like the mms6 DNA was not cut at all. Therefore is doubtful that the ligations will work.

June 9/2010

(in the lab: TF, JV)
Objective: Transform mms6-dT ligation reactions from June 8/2010.
Method: Followed transformation protocol and transformed the following:

mms6 (D9) + dT (C1)
mms6 (D10) + dT (C1)
pUC19 (positive control)
Water (negative control)

Results: No transformants on plates.

June 10/2010

(In the lab: AV, HB, JV)
Objective: Repeat ligation of mms6 (B9,D9,D10) and dT (C1).
Method: mms6 Restriction

2µL mms6 pDNA
2µL Red buffer
0.25µL EcoRI
0.25µL SpeI
15.5µL MilliQ H₂O

dT Restriction

2µL dT pDNA
2µL Orange buffer
0.25µL EcoRI
0.25µL XbaI
15.5µL MilliQ H₂O

Incubated for 1 hour at 37^oC.
Killed enzymes by heating to 80^oC for 20 minutes
Ligation

5µL dT pDNA
5µL mms6 pDNA
0.5µL T4 DNA Ligase
2µL 10x buffer
7.5µL MilliQ H₂O

Incubated at room temperature overnight.
Results:
Analyzed on 1% TAE agarose gel:

Lane	Sample	Load (µL)
1	1kb ladder	0.5 ladder; 2 dye; 9.5 MilliQ H₂O
2	Unrestricted mms6 (B9)	5 DNA; 2 dye; 5 MilliQ H₂O
3	Restricted mms6 (B9)	5 DNA; 2 dye; 5 MilliQ H₂O
4	Unrestricted mms6 (D9)	5 DNA; 2 dye; 5 MilliQ H₂O
5	Restricted mms6 (D9)	5 DNA; 2 dye; 5 MilliQ H₂O
6	Unrestricted mms6 (D10)	5 DNA; 2 dye; 5 MilliQ H₂O
7	Restricted mms6 (D10)	5 DNA; 2 dye; 5 MilliQ H₂O
8	Unrestricted dT (C1)	5 DNA; 2 dye; 5 MilliQ H₂O
9	Restricted dT (C1)	5 DNA; 2 dye; 5 MilliQ H₂O
10	D9 + C1 Ligation (June 8/2010)	5 DNA; 2 dye; 5 MilliQ H₂O

Gel run for 80 minutes at 100V

Looks like everything was restricted. Ligations may work this time.
Follow-up: Ligation mixtures can be restriction tested and (if cutting works) transformed into DH5α cells.

Objective:<b> Transform pDNA from distribution plates into DH5α cells to generate glycerol stocks for lab.

<b>Method:

Component	Volume (µL)
MilliQ H₂O	15.5
Buffer	2
pDNA	2
Enzyme	0.25 + 0.25