Monday - 6/24/13

Today was spent planning and troubleshooting the digest and ligation protocols as well as our alternative miniprep and gel purification method. I made a few modification to our current digest protocol and Joe showed me an alternative method to try tomorrow. I also wanted to attach amilCP to some previous promoter+RBS ligations, but did not plan that part out correctly and will have to redo that part tomorrow. Bill was responsible for all of the miniprep and gel purification experiments, and Alex came in to show us some new techniques that we might be able to incorporate into this process. Lastly, I did all of the day 1 preparations so that I can make a new batch of competent cells tomorrow using Alex’s protocol.

Restriction digest optimization experiments

Make changes to try and eliminate the background problem

Increase amount of DNA to 500 ng / 25 μL
Change buffer from CutSmart to NEBuffer#4
Do incubation cycle for 1 hr. for all parts

Choose 2 promoters + RBS + reporter + plasmid backbone to use

J23100 - 59.1 ng/μL (strong constitutive)
R0010 - 70.2 ng/μL (lac inducible & larger promoter)
B0034 - 89.7 ng/μL (standard RBS)
K592009 - 137.8 ng/μL (amilCP blue reporter)
pSB1C3 - 265.7 ng/μL (standard backbone for part submission)

	J23100	R0010		B0034	K592009		pSB1C3
DNA	8.46 μL	7.12 μL	DNA	5.57 μL	3.63 μL	DNA	1.88 μL
ddH2O	13.04 μL	14.38 μL	ddH2O	15.93 μL	17.87 μL	ddH2O	19.62 μL
NEBuffer #4 (10x)	2.50 μL	2.50 μL	NEBuffer #4 (10x)	2.50 μL	2.50 μL	NEBuffer #4 (10x)	2.50 μL
EcoRI	0.50 μL	0.50 μL	XbaI	0.50 μL	0.50 μL	EcoRI	0.50 μL
SpeI	0.50 μL	0.50 μL	PstI	0.50 μL	0.50 μL	PstI	0.50 μL

Prepare and sterilize items for competent cells

1x - 500 mL SOB
2x - Flat bottom centrifuge bottles

Ligation reactions

Add the following to a PCR tube for each reaction (2x)

(Note: Keep T4 DNA Ligase as cold as possible)

ddH2O - 4 μL
10x T4 DNA Ligase Buffer - 1.5 μL
T4 DNA Ligase - 0.5 μL
Upstream Insert - 4 μL (J23100, R0010)
Downstream Insert - 4 μL (B0034)
Backbone - 1 μL (pSB1C3)

Add the following to a PCR tube for each reaction (6x)

(Note: Keep T4 DNA Ligase as cold as possible)

ddH2O - 5 μL
10x T4 DNA Ligase Buffer - 1.5 μL
T4 DNA Ligase - 0.5 μL
Upstream Insert - 4 μL (P100+RBS, P108+RBS, P110+RBS, P114+RBS, P10+RBS, P11+RBS)
Downstream Insert - 4 μL (K592009)

(I didn’t plan these ones out correctly, so it probably won’t work. Perhaps we will some blue colonies if I get lucky. Use a different backbone next time)

Negative control

ddH2O - 12 μL
10x T4 DNA Ligase Buffer - 1.5 μL
T4 DNA Ligase - 0.5 μL
Backbone - 1 μL

Incubate in thermocycler

Overnight @ 16° C

Make overnights

TOP10 cells in SOB

Tuesday - 6/25/13

Today was really busy. I realized that I used backbone from a plasmid rather than the linearized PCR product for my experiments yesterday, so all of those reactions turned out to be a complete waste. I was able to redo 6 of the ligations today using the quick protocols, so we will see how that turns out tomorrow. I managed to do both the digest and ligation protocols with help from Bill on several steps while also making a new batch of competent cells. I was able to pre-chill the rotor overnight and also between the spin cycles, and also performed most of the protocol in the cold room, so hopefully these cells turn out really nicely. I will do the iGEM competent cell efficiency test on them tomorrow to find out. Meanwhile, Bill continued working on the gel extraction experiments. Lastly, we transformed all of the ligation products and with any luck we will come in tomorrow and see some blue colonies.

Make competent cells

See protocol in protocols folder

Pre-chilled rotor overnight and between spins
Performed all steps after growth in the cold room

Absorbance measurements

t (min)	0	45	75	90	105	120	135	150	155
OD	0.0583	0.0734	0.1145	0.1487	0.1955	0.2792	0.3616	0.4636	0.4848

Left at RT between last 2 measurements
Made (40x) 200 μL & (20x) 100 μL aliquots

NEB quick digest experiment - 500 ng / 50 μL

Mix the following in 0.2 mL PCR tubes

J23100 - 85.9 ng/μL
J23110 - 98.4 ng/μL
B0034 - 89.7 ng/μL
K592009 - 137.8 ng/μL
pSB1C3 - 63.9 ng/μL
pSB1A3 - 62.9 ng/μL

	J23100	J23110		B0034	K592009		pSB1C3	pSB1A3
DNA	5.82	5.08	DNA	5.57	3.63	DNA	7.82	7.95
ddH2O	37.18	37.92	ddH2O	37.43	39.37	ddH2O	35.18	35.05
NEB#4	2.50	2.50	NEB#4	2.50	2.50	NEB#4	2.50	2.50
EcoRI	0.50	0.50	XbaI	0.50	0.50	EcoRI	0.50	0.50
SpeI	0.50	0.50	PstI	0.50	0.50	PstI	0.50	0.50

All amounts in μL
Incubate in thermocycler

10 min. @ 37° C
20 min. @ 80° C

NEB quick ligation experiment - 20 μL reaction

Add the following to 0.2 mL PCR tubes

Promoter (2x)	2 μL	P_+RBS (6x)	2 μL
B0034	2 μL	K592009	2 μL
pSB1C3	2 μL	pSB1T3	2 μL
10x T4 Buffer	2 μL	10x T4 Buffer	2 μL
T4 DNA Ligase	1 μL	T4 DNA Ligase	1 μL
ddH2O	11 μL	ddH2O	11 μL

Incubate @ R.T. for 5 min.

2x - J23100, J23110
6x - P100+RBS, P108+RBS, P110+RBS, P114+RBS, P10+RBS, P11+RBS
2x (-) control - No inserts, +4 μL ddH2O

Transformations

See protocol in protocols folder

P100+RBS - quick prep
P110+RBS - quick prep
P100+RBS+amilCP
P108+RBS+amilCP
P110+RBS+amilCP
P114+RBS+amilCP
P10+RBS+amilCP
P11+RBS+amilCP
(-) control - chloramphenicol
(-) control - tetracycline

Wednesday - 6/26/13

When I came in today, no colonies had formed for any of the ligation reactions. Evidently one or both of the quick digest and ligation protocols were ineffective. A gel of the digest showed evidence that the digest may have worked to some extent, but was far from complete and this was definitely not conclusive. I decided to just set up a mass digest and ligation experiment to conduct on Thursday and Friday, so that we could transform on Friday and have our results on Monday. Lastly, I made up overnights for these experiments.

Competent cell efficiency test

Transform pUC19 of known concentration (5 pg/μL)

Old batch of cells

2x - 5 pg (1 μL) pUC19
2x - 10 pg (2 μL) pUC19

New batch of cells

2x - 5 pg (1 μL) pUC19
2x - 10 pg (2 μL) pUC19
1x - (-) control (cells only

Plate 20 μL

Run a gel on the quick digest products

1	2	3	4
Ladder	Dig. K592009	K592009 plasmid	Lin. pSB1T3

Forgot to dilute the K592009 plasmid

Make overnights for digest/ligation experiments

2x - J23100
2x - J23110
1x - R0010
3x - B0034
1x - pUC19

Thursday - 6/26/13

This morning the results of my competent cell efficiency test showed that the new batch of competent cells are better than the old batch, which was a good way to start the day. I tried out the Qiagen miniprep kit on my overnights, which produced mixed results but provided what I needed for my experiments. I performed a gauntlet of digest experiments today which will hopefully help to either eliminate or reduce the amount of background that is coming through in our ligations. The parameters tested were DNA concentration, choice of buffer, and incubation time. These digests also will provide the material for my ligation experiments tomorrow. I did one variation of that today with a somewhat proven ligation protocol to provide a basis for my digest experiments.

Competent cell efficiency results

Used 5 pg/μL pUC19

	Plate 1	Plate 2	Average	Efficiency
Old cells - 5 pg	8	5	6.5	1.57E7
Old cells - 10 pg	6	1	3.5	4.22E6
New cells - 5 pg	8	10	9	2.17E7
New cells - 10 pg	19	18	18.5	2.23E7

Miniprep of overnights using Qiagen kit

See protocol in protocol folder

On several of the tubes, the lysis solution did not turn yellow, while other did. Not sure why, but all appeared the same after neutralization
Spun down lysis 5 min. @ 13,200 rpm (3x)

5 minutes didn’t work so well, follow instructions next time

Combined minipreps of each part into 1 Eppendorf tube

J23100 - 243.1 ng/μL
J23110 - 223.6 ng/μL
R0010 - 67.7 ng/μL
B0034 - 82.0 ng/μL

Stored supernatant from pUC19 lysis @ -20° C for later experiment

Digest experiments to reduce background

Combine each of the following in 0.2 mL PCR tubes (25 μL)

250 ng	J23100 (A)	J23110 (B)	R0010 (C)	500 ng	J23100 (D)
DNA	1.03 μL	1.12 μL	3.69 μL	DNA	2.06 μL
ddH2O	20.47 μL	20.38 μL	17.81 μL	ddH2O	19.44 μL
NEBuffer #4	2.50 μL	2.50 μL	2.50 μL	NEBuffer #4	2.50 μL
EcoRI	0.50 μL	0.50 μL	0.50 μL	EcoRI	0.50 μL
SpeI	0.50 μL	0.50 μL	0.50 μL	SpeI	0.50 μL

250 ng	B0034 (E)	500 ng	B0034 (F)
DNA	3.05 μL	DNA	6.10 μL
ddH2O	18.45 μL	ddH2O	15.40 μL
NEBuffer #4	2.50 μL	NEBuffer #4	2.50 μL
XbaI	0.50 μL	XbaI	0.50 μL
PstI	0.50 μL	PstI	0.50 μL

250 ng	pSB1T3 (G - 5x)	500 ng	pSB1T3 (H)	250 ng	pSB1T3 (I - 2x)	250 ng	pSB1T3 (J - 2x)
DNA	3.97 μL	DNA	6.91 μL	DNA	3.45 μL	DNA	3.45 μL
ddH2O	17.53 μL	ddH2O	14.59 μL	ddH2O	18.05 μL	ddH2O	18.05 μL
NEBuffer #4	2.50 μL	NEBuffer #4	2.50 μL	NEBuffer #2	2.50 μL	Cutsmart	2.50 μL
EcoRI	0.50 μL	EcoRI	0.50 μL	EcoRI	0.50 μL	EcoRI	0.50 μL
PstI	0.50 μL	PstI	0.50 μL	PstI	0.50 μL	PstI	0.50 μL

Incubate in thermocycler

1 hr. @ 37°C, 20 min. @ 80°C (A, B, C, D, E, F, G, H, I - 2x, J - 2x)
15 min. @ 37°C, 20 min. @ 80°C (G’ - 2x)
2 hr. @ 37°C, 20 min. @ 80°C (G’’ - 2x)

Ligations for all digest experiments (15 μL)

Add each of the following to 0.2 mL PCR tubes

	1		2		3
A	2 μL	A	2 μL	A	2 μL
E	2 μL	E	2 μL	E	2 μL
G	1 μL	G’	1 μL	G’’	1 μL
Buffer	1.5 μL	Buffer	1.5 μL	Buffer	1.5 μL
T4 Ligase	0.5 μL	T4 Ligase	0.5 μL	T4 Ligase	0.5 μL
ddH2O	8 μL	ddH2O	8 μL	ddH2O	8 μL

	4 (2x)		5		6
D	2 μL	A	2 μL	A	2 μL
F	2 μL	E	2 μL	E	2 μL
H	1 μL	I	1 μL	J	1 μL
Buffer	1.5 μL	Buffer	1.5 μL	Buffer	1.5 μL
T4 Ligase	0.5 μL	T4 Ligase	0.5 μL	T4 Ligase	0.5 μL
ddH2O	8 μL	ddH2O	8 μL	ddH2O	8 μL

Negative control plates

	7 (2x)		8 (2x)		9 (2x)
G	1 μL	H	1 μL	I	1 μL
Buffer	1.5 μL	Buffer	1.5 μL	Buffer	1.5 μL
T4 Ligase	0.5 μL	T4 Ligase	0.5 μL	T4 Ligase	0.5 μL
ddH2O	12 μL	ddH2O	12 μL	ddH2O	12 μL

Negative control plates

	10 (2x)		11 (2x)		12 (2x)
J	1 μL	G’	1 μL	G’’	1 μL
Buffer	1.5 μL	Buffer	1.5 μL	Buffer	1.5 μL
T4 Ligase	0.5 μL	T4 Ligase	0.5 μL	T4 Ligase	0.5 μL
ddH2O	12 μL	ddH2O	12 μL	ddH2O	12 μL

Incubate overnight @ 16° C
Heat shock one of the #4’s for 20 min. @ 80° C in the morning (red mark)

Friday - 6/28/13

Today I conducted all of my ligation experiments, which were designed to test incubation temperature and time, ratio of insert to vector, and the effect of heat inactivation. Bill transformed all of the ligations for the digest experiments in the morning and I transformed all of the new ligation experiments in the afternoon. Bill used a few tetracycline plates to perform an additional experiment for the new batch of cells and realized we were then 2 plates short, so we had to make another batch of plates. Lastly, Bill made overnights for all of the plasmid backbones so that we could grow them over the weekend and perform a fluorescence test on Monday to see which vectors will express our proteins the most effectively.

Ligation experiments

Add the following to 0.2 mL PCR tubes

	13 - 2x		14 - 2x
D	1	B	2
F	1	F	1
H	0.5	H	0.5
T4 Buffer	1.5	T4 Buffer	1.5
T4 Ligase	0.5	T4 Ligase	0.5
ddH2O	10.5	ddH2O	9.5

Incubate for 30 min. @ R.T.
Heat inactivate 1 of each for 20 min. @ 80° C (red marks)

	15 - 2x		16 - 2x
D	1	B	2
F	1	F	1
H	0.5	H	0.5
T4 Buffer	1.5	T4 Buffer	1.5
T4 Ligase	0.5	T4 Ligase	0.5
ddH2O	10.5	ddH2O	9.5

Incubate for 1 hr. @ R.T.
Heat inactivate 1 of each for 20 min. @ 80° C (red marks)

	16 - 2x		17 - 2x
D	1	B	2
F	1	F	1
H	0.5	H	0.5
T4 Buffer	1.5	T4 Buffer	1.5
T4 Ligase	0.5	T4 Ligase	0.5
ddH2O	10.5	ddH2O	9.5

Incubate for 1 hr. @ 16° C
Heat inactivate 1 of each for 20 min. @ 80° C (red marks)

	19 - 2x	20 - 2x		21 - 2x
C	1	2	C	5
F	0.5	1	E	5
G	1	1	G	1
T4 Buffer	1.5	1.5	T4 Buffer	1.5
T4 Ligase	0.5	0.5	T4 Ligase	0.5
ddH2O	10.5	9	ddH2O	2

Incubate for 1 hr. @ 16° C
Heat inactivate 1 of each for 20 min. @ 80° C (red marks)

Transform all of the ligation products

Need 37 tetracycline plates