Week of 7/11 - 7/17

7/11

• Ran PCR on the construct (5x) 
• PCR'ed 84.2 ng/ml construct
• 5 replicates made using Phire Polymerase
• Used the following program for the thermocycler: 

Thermocycler ProgramStepTemperature (Degrees C)Duration (seconds)

Initial Denaturation	98	30
Denaturation	98	20
Annealing	66	5
Extension	72	30
Final Extension	72	60
Hold	4	Hold
After step 4, return to step 2. Repeat steps 2-4 thirty-five times before continuing to step 5 and on.

Water	19.5 uL
Phire Polymerase	25 uL
Template	0.5 uL
Forward Primer	2.5 uL
Reverse Primer	2.5 uL

• Restreaking of YEP352GAP transformed cells for miniprep
• Restreaking placed in incubator at 12am

7/12

• Made gel for electrophoresis of PCR construct 
• Thermocycler Program: Same as PCR from 7/11

Q5 Rx Buffer	10 uL
10nM dNTPs	1 uL
10 uM Forward Primer	2.5 uL
10 uM Reverse Primer	2.5 uL
Template (Q5 PCR Pw.1 for 6/30/16)	0.5 uL
Q5 Polymerase	2.5 uL
Water	33 uL

• LB liquid culture (3ml with 3ul of ampicillin)
• Loaded 10ul Ladder DNA.
• Loaded 1ul dye + 5ul PCR product
• Ran gel at 115V
• Checked gel ladder → faint, no band for the PCR product
• Made new gel, loaded with the same amount of reagents as before → ran at 90V → No ladder band
• Ran it again at 115V → visible ladder, no PCR band
• Preparing a gel to run ladder and construct that previously showed a strong band → used this to diagnose a problem with gel setup. Ran TDGF1 152.9 Phire Child. Lane 1 is ladder, Lane 2 is that construct (10ul) with 2ul of dye
• Setup new PCR using the 6/30/16 PCR construct

1. 98°C → 30sec
2. 98°C → 20sec
3. 66°C → 5sec
4. 72°C → 30sec
5. 72°C → 1min
6. 4°C → hold

7/13

• Ran 5ul of PCR with ladder, cancer construct and vaccine construct → to diagnose problem with gel
• Miniprep of Dean vector (3ml LB culture)
• Purification of PCR product → nanodropped 16.4ug/ul
• Nanodrop of:

1. Yellow 7/13 construct NEB purified → 16.4ng/ml
2. Blue gel purification → 48.7ng/ul
3. Pink PCR purification 7/13 → 102.2ng/ul
4. 7/13 -RK Vector 1 → 48.2ng/ul
5. 7/13 -RK Vector 2 → 96.3ng/ul

7/14

Project Progress

Ran a gel PCR of TDGF-1

Lanes:

1. Ladder
2. PCR1
3. PCR2
4. PCR3
5. Digest
6. Digest Control

PCR of TDGF-1 IDT Construct

• 3 reactions set up with the same setup as test 5 from 6/30
• Annealing Temperature at 66°C for 5 seconds

7/15

• Ran a gel on PCR'ed Construct
• 1st gel failed → Samara poured a gel onto Brian's gel
• All 3 PCR products worked → purified PCR → 30ul PCR product in each tube → 150ul building buffer

Nanodrop

PCR TubeConcentration (ug/ul)

PCR #1	15.6
PCR #2	12.3
PCR #3	11.6

• PCR IDT construct and 1 PCRed construct with 130.9 ug/ul concentration

7/17

• Epoch purification of one of PCR products from 7/14
• Same protocol at 7/15
• Approx. 10ul of DNA in tube for PCR use
• 30ul of elution buffer used
• Elution step centrifuged for 2 minutes at 13000RPM

Ran a Nanodrop

ContentConcentration (ug/ul)260/280

Sample I	22.2	1.79
Sample II	22.9	1.79

IDT Construct Digest (10ul Reaction)

ContentExperiment (ul)Control (ul)

H 2 O	3.5	4.5
10X Cutsmart	1	1
XhoI	.5	X
PvoII-HF	.5	X
DNA (22.2ug/ul)	4.5	4.5

YEP352GAP Vector Digest (10ul Reaction)

ContentExperiment (ul)Control (ul)

H 2 O	6.9	7.9
10X Cutsmart	1	1
XhoI	.5	X
PvoII-HF	.5	X
DNA (22.2ug/ul)	1.04	1.04

• Digested at 37°C in incubator for an hour
• 2ul of 6X Purple Loading Dye added to each of the four tubes after an hour digest to inactivate enzyme

Ran a gel

Lane:

1. Brian's Construct
2. Construct Digest - Control
3. Construct Digest - Experimental
4. Ladder
5. Vector Digest - Experiment
6. Vector Digest -Control

• Ran at 116V for 30 minutes