Week of 8/15 - 8/21

8/15

Gel of purified digests from 8/14

Lanes:

1. Epoch 1 Control
2. Epoch 1 Experimental
3. Ladder
4. Epoch 2 Experimental
5. Epoch 2 Control

PCR purified and nanodropped the digests

ContentConcentration (ng/ul)260/280

Epoch 1	9.2	2.26
Epoch 2	7.9	1.91

Qiagen 1 (Both enzymes)

ContentExperimental ulControl ul

H 2 O	18.85	19.85
Cutsmart	2.5	2.5
PvuII-HF	0.5	X
XhoI	0.5	X
DNA	2.65	2.65

Qiagen 1 (Sequential)

ContentExperimental ulControl ul

H 2 O	19.85	19.85
Cutsmart	2.5	2.5
XhoI	0.5	X
DNA	2.65	2.65

Qiagen 2 (Both enzymes)

ContentExperimental ulControl ul

H 2 O	19.07	20.07
Cutsmart	2.5	2.5
PvuII-HF	0.5	X
XhoI	0.5	X
DNA	2.43	2.43

Qiagen 2 (Sequential)

ContentExperimental ulControl ul

H 2 O	19.57	20.07
Cutsmart	2.5	2.5
Enzymes	0.5	X
DNA	2.43	2.43

498.9 ng/ul Plasmid

Both Enzymes

ContentExperimental ulControl ul

H 2 O	20.5	21.5
Cutsmart	2.5	2.5
PvuII	0.5	X
XhoI	0.5	X
DNA	1.0	1.0

Sequential

ContentExperimental ulControl ul

H 2 O	21	22
Cutsmart	2.5	2.5
Enzymes	0.5	X
DNA	1.0	1.0

Ran gels of plasmid digets

Gel 1

Lanes:

1. Qiagen 1 Control
2. Qiagen 1 (Both)
3. Qiagen 1 (Sequential)
4. Ladder
5. Qiagen 2 (Sequential)
6. Qiagen 2 (Both)
7. Qiagen 2 Control

Gel 2

Lanes:

1. Ladder
2. Old Sequenced
3. Old Both
4. Old Control

Ran gel of plasmids for gel extract

Lanes:

1. Ladder
2. Qiagen 1 Sequential
3. Qiagen 1 Both
4. Quaigen 2 Sequential
5. Qiagen 2 Both

Troubleshooting

Ran a gel and digest to diagnose if PvuII was denatured

205.6ng/ul

ContentExperimental ulControl ul

H 2 O	19.6	20.1
Cutsmart	2.5	2.5
PvuII	0.5	X
DNA	2.43	2.43

Gel of digests from the morning and PvuII-HF diagnostic

Lanes:

1. Qiagen 1 Control
2. Qiagen 1 Both
3. Qiagen 1 Sequential
4. Qiagen 2 Both
5. Qiagen 2 Sequential
6. Ladder
7. PvuII Diagnostic
8. PvuII Diagnostic Control

• Plasmid extracted from gel

Nanodrop of plasmid gel extract

ContentConcentration (ng/ul)260/280

Plasmid Gel Extract	7.1	1.45

8/16

Diagnostic PvuII-HF Digest

188.1 ng/ul

205.6 ng/ul

Ran a gel on PvuII Digest

Lanes:

1. Ladder
2. 205.6 Control
3. 205.6 Digest
4. 188.5 Control
5. 188.5 Digest

Nanodropped PvuII Digest

ContentConcentration (ng/ul)260/280

188.5	1.7	1.36
205.6	1.4	2.05

PCR of BioBrick Parts

J23119

Contentsul

Q5 MasterMix	12.5
H 2 O	6,5
10 uM F Primer	2.5
10 uM R Primer	2.5
Template	1

E0040

Contentsul

Q5 MasterMix	12.5
H 2 O	6,5
10 uM F Primer	2.5
10 uM R Primer	2.5
Template	1

BOO15

Contentsul

Q5 MasterMix	12.5
H 2 O	6,5
10 uM F Primer	2.5
10 uM R Primer	2.5
Template	1

8/17

Gel of old PEP352GAP plasmids to test viability + Gel of BioBrick PCRs

Gel #1

Lanes:

1. 498.4
2. 311.5
3. 259.0
4. 296.6
5. Ladder
6. 176.5
7. 277.3
8. 366.6

Gel #1

Lanes:

1. 133.4
2. 188.5 PvuII
3. 205.6 PvuII
4. 142.6
5. Ladder
6. J23119
7. E0040
8. B0015

• 142.6 was thrown out
• Gel #1 looked a lot better than #2

100ul Sequential digest - PvuII and then XhoI

498.4 ng/ul

366.6 ng/ul

• Incubated at 37°C for an hour

Nanodropped PvuII Digest

ContentConcentration (ng/ul)

498.4	14.2
366.6	29.6

XhoI Digest

29.6

• Incubated at 37°C for an hour

Ran a gel on sequential digests

Lanes:

1. Ladder
2. 29.6
3. 29.6
4. 14.2

Ligation of Dean Vector + Insert

Contentul

10X T4 Buffer	2.0
Vector	3.0
Insert	9.0
H 2 O	5.0
Ligase	1.0

8/18

8/19

Miniprep of Dean vector and BioBricks

• Four tubes of Dean vector
• Three tubes of BioBricks




Nanodrop of Miniprepped Dean Vectors + BioBricks

ContentConcentration (ng/ul)260/280

J23119	166.3	1.96
B0015	137.4	1.93
E0040	139.8	1.98
DV #1	365.5	1.92
DV #2	230.5	1.93
DV #3	282.3	1.94
DV #4	113.0	1.91

Initial Digests

J23119 - 166.3ng/ul

ContentExperimental ulControl ul

H 2 O	38	39
Cutsmart	5	5
EcoRI-HF	0.5	X
SpeI	0.5	X
DNA	6	6

J61100 - 1.85ng/ul

ContentExperimental ulControl ul

H 2 O	44	45
Cutsmart	5	5
XbaI	0.5	X
PstI	0.5	X
DNA	0.5	0.5

E0040 - 139.8/ul

ContentExperimental ulControl ul

H 2 O	36.9	37.9
Cutsmart	5	5
EcoRI-HF	0.5	X
SpeI	0.5	X
DNA	7.15	7.15

B0015- 137.4ng/ul

ContentExperimental ulControl ul

H 2 O	36.7	37.7
Cutsmart	5	5
XbaI	0.5	X
PstI	0.5	X
DNA	7.28	7.28

Gel of Digests

Gel #1

Lanes:

1. J23119 C
2. J23119 E
3. J23119 E #2
4. Ladder
5. E0040 E
6. E0040 E #2
7. E0040 C

Gel #2

Lanes:

1. J61100 C
2. J61100 E
3. J61100 E #2
4. Ladder
5. B0015 E
6. B0015 E #2
7. B0015 C

8/20

Digests of GFP, Promoter, and Terminator

Promoter

ContentExperimental ulControl ul

H 2 O	32	33
Cutsmart	5	5
EcoRI	0.5	X
SpeI	0.5	X
DNA	12	12

GFP

ContentExperimental ulControl ul

H 2 O	29.7	30.7
Cutsmart	5	5
EcoRI-HF	0.5	X
SpeI	0.5	X
DNA	14.3	14.3

Terminator

ContentExperimental ulControl ul

H 2 O	29.4	30.4
Cutsmart	5	5
XbaI	0.5	X
PstI	0.5	X
DNA	14.6	14.6

8/21

Nanodrop of BB Component MiniPreps

ContentConcentration (ng/ul)260/280

B0015	201.5	1.99
J23119	225.8	1.99
E0040	179.3	2.03