Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

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Contents

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

We made competent cells to use for transformations.

Procedure:

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: We received the synthesized TE33 antibody genes from IDT on plasmids. We transformed these plasmids into DH5a cells and plated on LB+amp the find transformants.

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

  1. 94 degrees 5 min
  2. 94 degrees 1 min
  3. 50 degrees 1 min
  4. 72 degrees 1 min
  5. GOTO 2. 29 cycles
  6. 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.

07-30-08 AbFrag Gel1.jpg


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

  1. 94 degrees 5 min
  2. 94 degrees 1 min
  3. 50 degrees 1 min
  4. 72 degrees 1 min
  5. GOTO 2. 29 cycles
  6. 72 degrees 5 min
  7. HOLD at 4 degrees

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

7 31 08 assembly.jpg


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

  1. 94 degrees 5 min
  2. 94 degrees 1 min
  3. annealing step 1 min
  4. 72 degrees 1 min
  5. GOTO 2. 29 cycles
  6. 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.

8 1 08 assembly gradient.jpg


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.


September 5, 2008

Flk-1 clones from 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 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
tube 15 16 17 18
0.5uL template flk link 2 flk link 2 flk link 3 flk link 3
A B A B
primers 1uL forward & reverse for all
mastermix 8.25uL for all
H2O 39.25uL for all

PCR program

  1. 94 degrees 4 min
  2. 94 degrees 1 min
  3. 50 degrees 1 min
  4. 72 degrees 3 min
  5. GOTO 2. 29 cycles
  6. 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


9 26 08 biobricks.jpg


9 26 08 biobricks (2).jpg

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

PCR program

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

September 27, 2008

  • 0.8% agarose gel run of products from yesterday, 200V for ~45 minutes


9 27 08.jpg
  • first 8 reactions may have worked but bands not great, assembly reactions did not work

September 28, 2008

0.8% gel run of yesterday's products, 150 V, 38 min, 200 V, 10 min

September 30, 2008

p1010 plasmid for biobicks cut out of plate 1003, well 4A; soacked in 5uL warm TE (50°C) for ~20 min, heat shocked at 50°C for 60s, placed on ice 2 min, 200uL LB added, incubated at 37°C, 1 hour, plated on LB + Amp and incubated overnight.

Restriction digest of our biobricks 18uL H2O 2uL 10x buffer Z 1uL EcoRI and PstI 1,2,4 uL DNA (H chain, L chain, SCA, flk)

p1010 transformatoin was stupid, transformed another plasmid instead, 1008, 1C

tube 1 2 3 4 5 6
template A=0.5uL B=1.0uL C=1.5uL (all flk-1 B)
primers 1uL forward & reverse for all
mastermix 8.25uL for all
H2O 39.25uL for A 38.75uL for B 38.25uL for C
tube 1 2 3 4 5 6
gradient 55.0 57.8 59.3 61.0 63.5 65.0
temps (1) (5) (6) (7) (9) (12)

Run for 39 cycles

October 2, 2008

PCR products run on 0.8% gel, 200V, ~40min Same results as always

pSB1A3 & pSB1A7 cut out of registry and transformed Plated on LB+Amp

October 3, 2008

PCR from yesterday may have kind of worked, just not a lot of product, running another gel to find out

Transformation failed, transforming w/ RDR plasmid to test transformation protocol

Hard to tell if PCR products are correct length, so-so bands

October 8, 2008

Transformations to get plasmids 1003:2F, 1008:10D, 1008:1C, 1008:6G

October 10, 2008

DNA purification using S prime PerfectPrep(TM) Spin Mini kit on transformed bacteria from 10/8/2008

October 14, 2008

Transformation using plasmid from 10/10/2008

October 16, 2008

Transformation failed, duh top 10 50uL cells transformed with 1003:10D, 1003:2F, plasmid from 10/10/2008, and pUC19 control

PCR to make ARTK fusion

tube 1 2 3 4 5
template 0.5uL SCA + 0.5uL flk-link3(8) 0.5uL SCA + 1.0uL flk-link3(8) 0.5uL SCA + 1.5uL flk-link3(8) 0.5uL SCA + 2.0uL flk-link3(8) 0.5uL SCA + 1.5uL flk-link3(8)
primers 1.0uL forward & reverse none
mastermix 20.0uL for all
H2O 27.0uL 26.5uL 26.0uL 25.5uL 28.0uL

October 17, 2008

Transformation failed. 1% gel of yesterday's PCR run, 200V, ~40min, no bands

October 18, 2008

Using phusion to get ARTK

tube 1 2 3 4 5
template 0.5uL SCA + 0.5uL flk-link3(8) 0.5uL SCA + 1.0uL flk-link3(8) 0.5uL SCA + 1.5uL flk-link3(8) 0.5uL SCA + 2.0uL flk-link3(8) 0.5uL SCA + 1.5uL flk-link3(8)
primers 1.0uL forward & reverse no primers
buffer 10.0uL for all
phu 0.5uL for all
H2O 36.5uL 36.0uL 35.5uL 35.0uL 37.5uL


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