Week of 7/25 - 7/31

                                            7/25

3X Phire Reactions (50ul)

Contentsul

Phire	25
H 2 O	23.2
50 uM F Primer	0.5
50 uM R Primer	0.5
95.1ug/ul Construct	0.8

• PCR run with 30 cycles and annealing temperature at 66°C

Run on a gel

• 10ul ladder
• 5ul each PCR reaction
• Ladder worked but lanes were smeared
• Construct was left out over the weekend, so it was probably degraded

Three PCR reactions

• Ran three PCRs with similar amounts of reagents as above, except each used a different concentration of template

                                           7/26

New 3 replicants of 50 ul PCR with Phire of IDT Construct

Contentsul

Phire	25
F Primer	1
R Primer	1
Template	1/0.75/0.5
H 2 O	22/22.25/22.25

• PCR was run with 30 cycles at normal settings with the annealing temperature at 66°C

Ran a gel on the 3x Phire PCR products

• Gel ended up looking good
• Nanodropped it but didn't get a good concentration

Ran PCR on 28.9 ng/ul and 10.98 ng/ul constructs (20ul reactions)

Ran nanodrops on the following

Contentng/ul260/280

Re-do of 28.9 ng/ul	29.1	1.9
Re-do of 10.8 ng/ul	10.8	Bad
7/26 Replicate of 10 ng/ul precursor	10.7	2.5
7/26 of 10 ng/ul child	16.8	2.06

Experiment

• Tried making another 10ng/ul child with longer PCR tubes
• Nanodropped to 30.1 ng/ul

                                              7/27

Ran a gel on the 8 PCR products

Lanes:

1. Ladder
2. PCR 1
3. PCR 2
4. PCR 3
5. PCR 4
6. PCR 5
7. PCR 6
8. PCR 7

• Ladder looked good but the PCR Products smeared

Ran a new gel including the digests + PCR product

Lanes:

1. 10 ul Ladder
2. PEP352GAP Experiment
3. Empty
4. PEP352GAP Control
5. Construct Experiment
6. Construct Control
7. Empty
8. PCR 8

Ran a PCR (50ul)

Contentul

Phire	25
100 uM Fwd Primer	0.25
100 uM Rev Primer	0.25
10.8 ng/ul Template	1
H 2 O	23.5

PCR Settings at 35 cycles:

PhaseTemperature (°C)Time (sec)

Initial Denaturation	98	60
Denaturation	98	25
Annealing	66	20
Extension	72	45
Final Extension	72	60

• Labelled "7/27 last hope CR-1 PCR"
• Gel looked good
• PCR purified + nanodropped → 14.6 ng/ul

                                              7/28

PCR of 10.8 ng/ul Construct Sample

Contentul

Phire	25
50uM F	0.5
50 uM R	0.5
10.8 un/ul Template	2
H 2 O	22

PCR Settings: 35 Cycles

PhaseTemperature (°C)Time (sec)

Initial Denaturation	98	30
Denaturation	98	20
Annealing	66	10
Extension	72	30
Final Extension	72	60

Digest: Mimicked Protocol from 7/18

• PPEP352GAP : 498.4 ng/ul
• Construct - 28.9 ng/ul

50 ul Reaction

ContentExperimental (ul)Control (ul)

Water	42	43
10x Cutsmart	5	5
XnoI	.5	X
PvoII-HF	.5	X
498.4 ng/ul DNA	2	2

25 ul Reaction

ContentExperimental (ul)Control (ul)

Water	4.2	5.2
10x Cutsmart	2.5	2.
XnoI	.5	X
PvoII-HF	.5	X
498.4 ng/ul DNA	17.3	17.3

• Mixed reactions by flicking tube
• Spin down in touches to mix
• Incubate in incubator 37°C for an hour

Running gel of PCR Reaction + Digest

Lanes:

1. PCR Reaction
2. Construct Control
3. Construct Digest
4. Ladder
5. Vector Digest
6. Vector Control

• Purified digest using Epoch Kit

Nanodrop of Digests

ContentConcentration (ng/ul)260/280


Construct	14.6	1.85
Vector	14.6	1.80

20 ul Ligation of our hopes and dreams

Contentul

10X T4 Buffer	2
Vector	4 (58.4 ng)
Construct	1.55 (22.69 ng)
H 2 O	11.45
Ligase	1

• Gently Mixed Reaction
• Incubate at room temperature 2 hours
• Chill on ice and put in freezer

Transformations

• Transformed Emmanuel + Ryan's ligations, control + piggybac's
• 1:10 dilution made of each transformation
• Created LB Cultures
• Incubated
• PUC19 Control grown on chloramp plates
• Did not survive

                               7/29

• LB Cultures (8ml each) setpup for Ryan's Ligation, Emmanuel's Ligation, PiggyBac

                               7/30

Miniprep of Ligated + Transformed vector cultures

• Spun at 4000rpm for 5 minutes at 23°C
• Spun an additional 2 minutes at 4000rpm

Mixed Epoch McI Buffer into Qiagen Protocol

• 250ul of MXI used for 8ml LB Preps
• Lysis Rxn Sitting fo 4 minutes
• Eluted with 2 step of 2ul nuclease free NEB Water

Nanodropped miniprepped plasmids - Water as background

ContentConcentration (ng/ul)260/280Graph Character

PiggyBac	121.2	1.91	Lit
PiggyBac 1:10	423.7	1.87	Lit
Emmanuel	193	1.92	Lit
Ryan	446.4	1.89	Lit
Ryan 1	226.3	1.94	Lit
Ryan 2	232.2	1.93	Lit
Ryan 3	272.6	1.90	Lit
Ryan 4	335.5	1.84	Lit

Running gels with miniprepped plasmids from earlier today

Lanes:

1. PiggyBac: 5ul + 1 ul dye
2. PiggyBac: 2.5 ul + 1 dye
3. Ladder
4. Ryan: 2.5 ul + 1 ul dye
5. Ryan 1: 4.17ul + .83 ul dye
6. Ryan 2: 4.17ul + .83 ul dye
7. Ryan 3: 4.17ul + .83 ul dye
8. Ryan 4: 4.17ul + .83 ul dye

• Ran at 116V for 65 minutes
• Gel looked strange

Prepped digest of Lanes 1, 2, 4 and 7

• XbaI, EcoRI, XhoI and PvoII-HF respectively

Lane 1: PIggyBac - 121.2 ng/ul

ContentExperimental ulControl ul

H 2 O	35.75	36.75
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	8.25	8.25

Lane 2: PIggyBac 1:10 - 423.7 ng/ul

ContentExperimental ulControl ul

H 2 O	41,6	42.6
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	2.36	2.36

Lane 4: Ryan - 446.4 ng/ul

ContentExperimental ulControl ul

H 2 O	41.8	42.8
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	2.24	2.24

Lane 7: Ryan 3 - 272.6 ng/ul

ContentExperimental ulControl ul

H 2 O	40.3	41.3
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	3.67	3.67

Ran gel of digest

• Added 10ul of dye to each tube to stop reaction
• Ran at 116V for 35 minutes

Gel #1

Lanes:

1. PiggyBac Control
2. PiggyBac Experimental
3. Ladder
4. PiggyBac 1:10 Experimental
5. PiggyBac 1:10 Control

Gel #2

Lanes:

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

                                7/31

Digest of Ryan and PB 1:10

• Redid the digest
• Miniprepped with phosphatase treatment for the digest
• Used 5 units/ul of Antarctic Phosphatase

Lane 2: PIggyBac 1:10 - 423.7 ng/ul

ContentExperimental ulControl ul

H 2 O	41,6	42.6
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	2.36	2.36

Lane 4: Ryan - 446.4 ng/ul

ContentExperimental ulControl ul

H 2 O	41.8	42.8
Cutsmart	5	5
EcoRI-HF	0.5	X
XbaI	0.5	X
DNA	2.24	2.24

Post digest procedures

• Flicked tubes and spun down in touches
• Incubated at 37°C for an hour
• Purified with Epoch kit
• Phosphatase and heat inactivated at 80°C for 2 minutes
• 4ul of phosphate buffer, 1 ul of phosphatase and 5ul of nuclease free H
  2
  O used

Ran a gel

• Used 8ul loading dye in each tube

Lanes:

1. PB Control
2. PB Experimental
3. Ladder
4. Ryan Experimental
5. Ryan Control

Ran a digest of Dean vector with various enzymes control

• 25ul reaction - 498.ng/ul

Xhol

Contentl ul

H 2 O	21
Cutsmart	2.5
XhoI	0.5
DNA	1

PvoII-HF

Contentul

H 2 O	21
Cutsmart	2.5
PvoII	0.5
DNA	1

No Enzyme - Control

Contentul

H 2 O	21.5
Cutsmart	2.5
DNA	1

Both Enzymes

ContentExperimental ul

H 2 O	20.5
Cutsmart	2.5
XhoI	0.5
PvoII-HF	0.5
DNA	1

Ran a gel

Lanes:

1. XhoI
2. PvoII-HF
3. Ladder
4. Both Enzymes
5. No enzyme

New 25ul Digest of Ryan and PB 1:10 miniprep

PB1:10 (423.7ng/ul)

ContentExperimental (ul)Control (ul)

H 2 O	20.3	21.3
Cutsmart	2.5	2.5
XbaI	0.5	X
EcoRI-HF	0.5	X
DNA	1.18	1.18

Ryan (446.4 ng/ul)

ContentExperimental (ul)Control (ul)

H 2 O	20.4	21.4
Cutsmart	2.5	2.5
XbaI	0.5	X
EcoRI-HF	0.5	X
DNA	1.12	1.12

Gel run with 10ul of each

Lanes:

1. PB Control
2. PB Experimental
3. Ladder
4. Ryan Experimental
5. Ryan Control