Week of 7/18 - 7/24

7/18

Ran a digest of PEP352GAP vector

ContentsExperimental (ul)Control (ul)

H 2 O	19.9	20.9
10x Cutsmart	2.5	2.5
XnoI	.5	X
PvoII-GF	.5 ul	X
DNA (306.6ug/ul)	1.63	1.63

• Mix tubes by flicking a few times
• Spin down in centrifuge
• Reactions stopped by adding 4.16 ul of purple loading dye to tubes

Running Gel

Lane:

1. Ladder
2. Vector Digest - Experimental
3. Vector Digest - Control

After gel run:

• Excised Vector Digest - Experimental
• Gel purified → 180mg of gel → 720 ul of dissolving buffer
• Ran Nanodrop

NanodropConcentration (ug/ul)260/280

Vector Digest	20.4	2.47
Construct Digest	2.3	-7.40

Ran gel on a digestion of vector + construct with ladder

Lane:

1. Construct Control
2. Construct Experiment
3. Ladder
4. Vector Experiment
5. Vector Control

• Excised Lane 2 (Construct Experiment) and put in fridge

7/19

Digest of Dean Vector (25ul Reaction)

ContentsExperimental (ul)Control (ul)

H 2 O	17.80	18.80
10x Cutsmart	1	1
XhoI	.5	X
PvoII-HF	.5	X
DNA (96.3ug/ul)	5.19	5.19

• Incubated at 37°C for 1hr. Purple loading dye, no SDS added (4.16ul)
• Restreaked Dean vector colony
• Gel extraction of construct digest excise
• 200mg of gel → 800ul of dissolving buffer
• Dissolved at 47°C
• Also made a new 10X TAE buffer

Ran a Nanodrop

ContentsConcentration (ug/ul)

YEP352GAP Miniprep - 7/13	31.7
YEP352GAP Miniprep - 7/12	10.8
Purification PCR Construct 1 - 7/15	24.0
Purification of PCR Construct 2 - 7/15	50.4

PCR of CR-1 PCR Product - Main Direct Program 1 (2 replicates - 50ul Reaction):

ContentsAmount (ul)

H 2 O	19
Phire	25
10uM F Primer	2.5
10uM R Primer	2.5
Template	1

Digest of PEP352GAP

ContentExperimental (ul)Control (ul)

H 2 O	11.6	12.6
10X Cutsmart	2.5	2.5
XhoI	.5	X
PvoII-HF	.5	X
DNA(50.4 ug/ul)	9.92	9.92

Ran a gel

Lane:

1. Ladder
2. PCR Tube #1
3. PCR Tube #2
4. Digest Vector - Experiment
5. Digest Vector - Control

Ran a second gel

• Ran with the same setup as the gel above
• Only PCR Tube #1 worked
• Gel purified and PCR purified

Ran a Nanodrop

ContentsConcentration (ug/ul)

PCR Purified - 7/19	95.1
Gel Purified PCR Product - 7/19	5.7

7/20

Q5 Master Mix (2X) PCR of construct - 2 Reactions (25ul)

ContentRx #1 (ul)Rx #2 (ul)

Q5 Master Mix	12.5	12.5
10mM F Primer	1.25	1.25
10mM R Primer	1.25	1.25
Template (95.1ug/ul)	.5	1
Milli-Q H 2 O	9.5	9.0

PCR Setup

• Ran for 35 cycles
• Lid heated to 105°C
• Preheated to 98°C
Phase°CTime (sec)

Initial Denaturation	98	30
Denaturation	98	10
Annealing	67	30
Extension	72	30
Final Extension	72	120
Hold	4	Held

• 2-3 mL LB cultures of YEP352GAP
  • e. coli prepped from each
  • Restreaking from 7/18
• 3mL of LB Broth with 3mL Ampicillin
• Control set up with RFP Plasmid e. coli
• Incubated at 37°C at 3:12am - 220rpm

Gel run on 7/20 Q5 PCR Product

Lane:

1. Ladder
2. Rx #1 5ul + 1ul dye
3. Rx #2 5ul + 1ul dye

Q5 PCR of PCR Template (Phire - 25ul Rx)

ContentsAmount (ul)

H 2 O	9.5
Master Mix	12.5
10uM F Primer	1.25
10uM R Primer	1.25
Template (95.1ug/ul)	.5

• Thermocycler set up to the same specifications as earlier today, except the annealing temperature is now 68°C
• Prepped a gel for running PCR Reactions

7/21

Miniprep of Dean Vector using 2-3ml

• LB culture made 7/20

1. Centrifuged at 4500rpm for 2 minutes and 30 seconds
2. Centrifuged at 4500rpm for 5 minutes, lysed LB cultures

• Followed NEB protocol from then on

Two gel runs

• Ran two consecutive gels with Reactions 1 and 2
• Gels ended up smearing
  • Make a serial dilution of template
  • Too many cycles (Maybe lower it to 25-30)
  • Primer concentration not optimal

3 Reactions set up with Q5 2X Master Mix Polymerase (25ul Rx)

ContentRx #1Rx #2Rx #3

H 2 O	9.9	9.7	9.5
10X uM F Primer	1.25	1.25	1.25
10X uM R Primer	1.25	1.25	1.25
Q5 Master Mix	12.5	12.5	12.5
95.1 ug/ul Template	0.1	0.3	0.5

• Machine set up normally except the annealing temperature set up for 67°C
• Ran at 28 cycles

7/22

Set up PCR Rx for construct (25ul Rx x2 - 28 cycles)

ContentRx1 (ul)Rx2 (ul)

H 2 O	9.5	9.9
10uM F Primer	1.25	1.25
10uM R Primer	1.25	1.25
95.1 ug/ul Template	0.5	0.1
Q5 Master Mix	12.5	12.5

• PCR run at normal settings except the annealing temperature is now 52°C