Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

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July 1, 2008

Made Yeast Media

YPD Medium, per liter:

10g yeast extract

20g peptone

20g dextrose

20g agar(only for plates)

1. Dextrose filter sterilized, the rest autoclaved

2. Weigh nutrients into flask double the volume you want to make, and stir to dissolve

3. Dextrose added to autoclaved media to equivalent of 20 g/L

4. Liquid media placed on bench, plate media placed in 65 degree water bath approximately 5 minutes

5. Poured into plates and allowed to solidify

July 17, 2008

E.Coli Media

LB medium, per liter

10g tryptone

5g yeast extract

5g NaCl

1 mL 1N NaOH

15g (agar for plates)

1. Antibody DNA resuspended in TE buffer, 0.1 μg/μL

2. 5mL LB inoculated with single colony DH5a pro

3. Incubated at 37 degrees overnight

July 18, 2008

Competent cells:

1. 3mL overnight culture of DH5a pro

2. Inoculated into 35mL of LB

3. OD600 checked, want 0.2-0.3

4. Place culture on ice for 3 minutes

5. Spin at 10,000 rpm for 7 minutes, discard supernatant

6. Resuspend in 10 mL cold 30 mM CaCl2, incubated on ice overnight

7. Glycerol added to 10%, cells in freezer

July 19, 2008

Transformation:

1. Cells put in ice 30 min with 1μL plasmid(100ng)

2. Heat Shock for 2 minutes at 42 degrees

3. Ice for 8 minutes

4. Add cells to 1ml LB

5. Grow for 1 hour

6. Plate 100 to 200 μL on LB and amp, grow overnight

July 21, 2008

Observations:

Plenty of colonies on all plates

Also, making 5mL overnight cultures for mini-prep tomorrow.

July 22, 2008

Mini spin prep on LC and HC colonies

Place DNA in 100μL H2O in freezer

July 29, 2008

All primers brought to standard concentration, 30mM

33.3μL H2O added per n mole of primer

July 30, 2008

Assembly PCR: amplify antibody gene fragments and then assemble them, light and heavy chains

tube	1	2	3	4	5	6	7	8
Antibody chain	H	H	L	L	H	H	L	L
DNA source	mini-prep				synthesized IDT genes
template	0.5ul
primers	1ul of appropriate forward and reverse primer
MgCl2	3ul	5ul	3ul	5ul	3ul	5ul	3ul	5ul
master mix	20ul
H20	to 50ul total volume

Master mix already contains MgCl2; should have added extra to some tubes.

PCR program

94 degrees 5 min
94 degrees 1 min
50 degrees 1 min
72 degrees 1 min
GOTO 2. 29 cycles
HOLD at 4 degrees

1.5% agarose gel made, 0.75g agarose in 50ml 0.5X TBE w/10ul EtBr.

20ul of PCR produts loaded on gel w/4ul 6X loading buffer, run at 200V for about half an hour.

All lanes had lots of DNA at ~375bp, PCR worked.

July 31, 2008

PCR to asemble fragments from yesterday.

tube	1	2	3	4
template from 7-30 PCR	2+3	2+7	6+7	1+8
template	0.5ul of both heavy and light chains
primers	1ul of appropriate forward and reverse primer
MgCl2	0ul	0ul	3ul	3ul
master mix	20ul
H20	to 50ul total volume

PCR program

94 degrees 5 min
94 degrees 1 min
50 degrees 1 min
72 degrees 1 min
GOTO 2. 29 cycles
72 degrees 5 min
HOLD at 4 degrees

Products run on a 1.5% agarose gel, no products of 700-800bp.

August 1, 2008

PCR troubleshooting of yesterday's reaction: use gradient for annealing temp (40-65 degrees), use more template, leave out end primers

Four series of reactions using a gradient annealing temp were run in addition to a fifth series omitting the end primers.

reaction series	A	B	C	D	E
template (5+7) from 7-30 PCR	0.5ul	1ul	1.5ul	2ul	1ul
primers	1ul of forward and reverse primers (omitted in E series)
master mix	20ul
H20	to 50ul total volume

annealing temps: 40, 43.8, 50, 54, 60, and 64 degrees

PCR program

94 degrees 5 min
94 degrees 1 min
annealing step 1 min
72 degrees 1 min
GOTO 2. 29 cycles
HOLD at 4 degrees

PCR products run on 1.5% agarose gel at 200V for ~20 minutes, then 240V for ~20 minutes.

Products in the 700-800bp range in all lanes. Perhaps it worked because of using a differnt product from the 7-30 PCR.

August 6, 2008

1.5% agarose gel of 8-1 PCR products run to cut out bands. Products A1, B1, C1, and E1 used.

Bands around 800bp cut out for all lanes and invitrogen gel extraction kit used to collect DNA in 50ul H2O.

Flk-1 clones from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=MGC-18600&Template=mgcMouseClones ATCC] streaked onto LB+amp and incubated at 37 degrees.

September 6, 2008

Colonies picked from flk-1 plate and inoculated into 5ml LB+amp, grown at 37 degrees.

Freeze-dried e. coli with YCp50-poly from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=87555&Template=vectors ATCC] resuspended in 5ml LB, grown at 37 degrees.

September 7, 2008

Mini spin prep of flk-1 and YCp50-poly cells, plasmids stored in 50ul H2O.

September 25, 2008

Biobricks

Bricks to be made: H chain, L chain, SCA, flk-1

tube	1	2	3	4	5	6	7	8
0.5uL template	H chain	L chain	SCA	SCA	SCA	SCA	flk-1	flk-1
	IDT	IDT	A	B	C	E	A	B
primers	1uL forward & reverse for all
mastermix	8.25uL for all
H2O	39.25uL for all

tube	9	10	11	12	13	14
0.5uL template	SCA w/ link	SCA w/ link	SCA w/ link	SCA w/ link	flk link 1	flk link 1
	A	B	C	E	A	B
primers	1uL forward & reverse for all
mastermix	8.25uL for all
H2O	39.25uL for all

PCR program

94 degrees 4 min
94 degrees 1 min
50 degrees 1 min
72 degrees 3 min
GOTO 2. 29 cycles
HOLD at 4 degrees

September 26, 2008

product from yesterday run on 1% gel, 200V for ~30 minutes
mostly good results, small amound of DNA for flk-1
lanes 1,2,6,8,12,14,16,18 cut out and purified with invitrogen gel extraction kit, eluted in 50ul H2O

PCR to assemble SCA-flk-1 fusion and increase flk-1 yield

tube	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
template	0.5ul flk-1 A	0.5ul flk-1 B	0.5ul flk link 1 A	0.5ul flk link 1 B	0.5ul flk link 2 A	0.5ul flk link 2 B	0.5ul flk link 3 A	0.5ul flk link 3 B	0.5ul SCA link + 0.5ul flk link 1	0.5ul SCA link + 0.5ul flk link 1 (crude)	0.5ul SCA link + 2ul flk link 1	0.5ul SCA link + 2ul flk link 1 (crude)	0.5ul SCA link + 0.5ul flk link 2	0.5ul SCA link + 0.5ul flk link 2 (crude)	0.5ul SCA link + 2ul flk link 2	0.5ul SCA link + 2ul flk link 2 (crude	0.5ul SCA link + 0.5ul flk link 3	0.5ul SCA link + 0.5ul flk link 3 (crude)	0.5ul SCA link + 2ul flk link 3	0.5ul SCA link + 2ul flk link 3 (crude)
primers	1uL forward & reverse for all
mastermix	8.25uL for all
H2O	to 50ul total volume