Team:Illinois/Antibody GPCR Fusion Notebook

Recipes

 * Tris-Cl, 1M
 * Dissolve 121g Tris base in 800ml H2O
 * Adjust to desired pH with concentrated HCl
 * Mix and add H2O to 1 liter
 * (Approximately, 20ml HCl for pH 7.4 and 42ml for pH 8.0)


 * EDTA, 0.5M (pH 8.0)
 * Dissolve 186.1g Na2 EDTA-2H2O in 700ml H20
 * Adjust pH to 8.0 with 10M NaOH(~50ml)
 * Add H2O to 1 liter


 * Breaking buffer - 100ml
 * 2ml Triton X-100
 * 1ml Sodium dodecyl sulfate (SDS)
 * 0.5844g NaCl (100mM)
 * 1ml 1M Tris-Cl pH 8.0 (10mM)
 * 200uL 0.5M EDTA (1mM)

Primers
PGK promoter Fwd:5'TTTT GAATTC AAAGATGCCGATTTGGGCGC Rev:5'TTTT GAGCTC GTTTTATATTTGTTGTAAAA

PGK terminator Fwd:5'TTAT GGGCCC GAAATAAATTGAATTGAATT Rev:5'TTTTG AAGCTT CAGCTTTAACGAACGCAGA

Ste2 Fwd:5'GCCC TCTAGA ATGTCTGATGCGGCTCCTTC Rev:5'TTAT GGGCCC TCATAAATTATTATTATCTT

Fus1 upstream Fwd:5'GTGG GAATTC TAATAATCAGAACTCCAACA Rev:5'GGCG TCTAGA TTTGATTTTCAGAAACTTGA

Fus1 downstream: Fwd:5'GCGA GGTACC TGAAAATAATATTGACGTTC Rev:5'TTAT GCGGCCGC TATTCACCAGACCCGCTCCT

22nd July

 * Yeast obtained from Dr. Zhao
 * W303a S. Cerevisiae

24th July

 * Prepared liquid culture for DNA extraction
 * Made 1M Tris. Cl pH 8.0
 * Made 4M ammonium acetate

22nd August

 * Attempted DNA extraction of W303a genomic DNA
 * Protocol from Wiley's Current Protocols in Molecular Biology
 * Result: Failed to finish protocol
 * Obtained more yeast from Dr. Zhao
 * W303a S. cerevisiae

25th August

 * Prepared overnight culture for DNA extraction (3:27pm)

26th August

 * Attempted DNA extraction
 * Prepped overnight culture

27th August

 * Performed PCR: PGK Terminator


 * PCR program:
 * 4 min 94 degrees
 * 25-30x 30s 94 degrees
 * 30s Tm primers
 * 1 min/KB 72 degrees
 * 7 min 72 degrees


 * For the gel: 5uL loading dye gel is in cold room


 * Prepped 3 overnight cultures

28th August

 * Extracted DNA from 4 cultures
 * Ran gel of PCR products (1.5% agarose, 200V)
 * Result: No bands present

2nd September

 * PCR: PGK Terminator

3rd September

 * Ran gel
 * Ladder lane 7
 * Sample 7 spilled
 * 1% agarose
 * 120V
 * Too low
 * 50 minutes
 * Poor results--no bands present

8th September

 * PCR: PGK Terminator


 * Prepped 4 overnight cultures
 * Yeast dried out again

9th September

 * Signs of life in 3 of the cultures
 * Wait until tomorrow
 * Ran gel on PCR from 8th September--no bands present
 * 150V, 50 minutes
 * No sign of DNA
 * Ladder from Courtney

10th September

 * Split culture
 * Ran gel from 8th September again--no bands present
 * 150V, 50 minutes
 * 0.75% gel
 * Ladder from Courtney

11th September

 * PCR: PGK Terminator

12th September

 * Isolated genomic DNA from 8 cultures of W303a yeast cells
 * Protocol from Wiley's Current Protocols in Molecular Biology
 * Labeled templates 1,2,3,4 and 1a,2a,3a,4a


 * Ran gel of PCR from 11th September
 * 1% agarose
 * 150V
 * 38 mins
 * Poor results

15th September

 * PCR PGK Promotor
 * Finnzymes Phusion High Fidelity DNA Polymerase
 * F-530, 20V (2V/uL)

18th September

 * Ran reaction mentioned on 15th September
 * Extracted DNA from gel from 8th September (PGK Terminator)

19th September

 * Gel of PGK Promotor from 15th September has no DNA present

23rd September

 * PCR: Fus1 Downstream
 * EPICENTRE Bioetechnologies - MasterAmp(TM) Taq DNA Polymerase


 * Per 50uL of reaction,


 * PCR Settings:
 * 4mins, 94 degree celcius
 * 30s, 94 degree celcius
 * 30s, 5 degrees below primer melting temperature
 * 1 min, 72 degree celcius -- to step 2 -- 30x
 * 7 min, 72 degree celcius

24th September

 * Ran gel of Fus1 Downstream from 23rd September
 * Result: No DNA present on gel

25th September

 * PCR: Fus1 Upstream


 * same protocol as 23rd September
 * The master mix contains the buffer, dNTPs, MgCl, and Taq
 * Template 3 (3 reactions run)

30th September

 * PCR: Fus1 Upstream


 * same protocol as 23rd September
 * Template 4 and 1a (3 reactions each)


 * Gel: 1% agarose, 150V, 35 minutes -> poor results
 * lanes 2,3 -> faint smear

1st October

 * PCR: Ste2
 * same protocol as 23rd September
 * Templates 2a, 3a, 4a used (3 reactions each)

3rd October

 * Ran Ste2 gel from 1st October

8th October

 * PCR: PGK Terminator


 * same protocol as 23rd September
 * Use DNA extracted from gel on 18th September (5 reactions)
 * Also extracted DNA from gel from 30th September (Fus1 Upstream)

9th October

 * PCR: Ste2
 * Protocol is the same as 23rd September
 * Template used is product from 1st October (9 reactions total)


 * PCR: Fus1 Upstream
 * Protocal matches 23rd September
 * Template used was DNA extracted from the gel from the 8th October, which came from the PCR run on 30th September (5 reactions total)


 * Ran Ste2 gel from today
 * Ran PGK terminator gel from yesterday

12th October

 * PCR: Fus1 (3 reactions each)
 * Template is products from 9th October

13th October

 * Ran gel of PCR with Fus1
 * Used 25uL or product, 5uL of loading dye

14th October

 * Fus1 PCR
 * Template is reaction from 12th October

15th October

 * PCR PGK Terminator


 * Ran gel of Ste2 from 10/14
 * Ran gel of Fus1 upstream from 10/14 (no ladder, oops)

16th October

 * PCR: Fus1 Downstream, PGK Promoter


 * Tube 1: Template 4 - Fus1
 * Tube 2: Template 4a - Fus1
 * Tube 3: Template 4 - PGK Promoter
 * Tube 4: Template 4a - PGK Promoter


 * ran Gel of above


 * ran Gel of PGK terminator from 10/15


 * Extracted Ste2 from 10/15 and re-amplified:

17th October

 * Ran gel of Ste2 from 10/16
 * Tube 1 - lane 2,3
 * Tube 2 - lane 5,6


 * PCR: Fus1 upstream
 * template is products from 10/14


 * PCR: PGK Terminator


 * Template is products from 10/15


 * Extracted Ste2(Gel from today)

18th October
Ran gel of Fus1 upstream and PGK terminator from yesterday

20th October

 * Extracting chromosomal DNA from yeast cells
 * W303A - Yellow
 * YPD DL
 * YHP1 YPD HD - One is orange (YHP1)(Cap was removed in incubator), Other is Yellow


 * Orange - YHP1 YPD HD
 * Green - YHP2 YPD HD
 * Pink - W303A YPA DL
 * Yellow - WD303A YPD DL

21st October

 * PCR: Ste2


 * 2 tubes
 * Block B of black machine
 * Annealing temperature: 31 degrees celcius


 * Ran gel on Ste2 from today

22nd October

 * New primers for biobricks are here
 * Brought to standard concentration, 30uM
 * Added 33.3uL of water per nmol primer


 * PCR: Fus1 and PGK Terminator


 * Forward and Reverse primers are new biobrick primers that arrived today
 * Template is PCR product from 17th October


 * A,B,C -> Fus1 Upstream -> 1,2,3
 * 1,2 -> PGK Terminator -> 4,5
 * Annealing temperature: 38 degrees celcius
 * In freezer in yellow case


 * PCR: Ste2


 * Forward primer, Reverse Primer are new primers that arrived today
 * Template is PCR product from 21st October
 * Two tubes in block B


 * The 3 gels in the cold room do not have EtBr

23rd October

 * Ran gel of Fus1 Upstream, PGK Terminator, Ste2 from yesterday
 * 1% Agarose


 * PCR: Fus1 Downstream, PGK Promoter


 * 4 reactions each of Fus1 Upstream(1,2,3,4) and PGK Promoter(A,B,C,D)
 * Gel shows no bands.


 * PCR: Fus1 Downstream, Fus1 Upstream, PGK Promoter, PGK Terminator, Ste2
 * Template genomic DNA from 20th October (x4 different reactions)
 * Used all Template


 * Gel shows no bands

24th October

 * Ran gel of Fus1 upstream(lanes 3,4,5), PGK terminator(lanes6,7), and Ste2(lanes 8,9) from 10/22
 * ladder is lane 2; 1 and 10 are nothing


 * PCR of Fus1 downstream, Fus1 upstream, PGK promoter, PGK terminator, Ste2
 * The templates were the rest of the genomic DNA extracts from 9/12
 * Three reactions of each gene were run


 * Gel from above:
 * (on left)Fus1 downstream lanes 2,3,4; Fus1 upstream lanes 5,6,7; PGK promoter lanes 8,9,10;
 * (on right)PGK terminator lanes2,3,10; Ste2 lanes 4,5,6; Fus1 downstream lanes 7,8,9;

25th October

 * Extracted DNA from the gel from 10/24
 * FUS1 upstream from the higher bands of lanes 3 and 4
 * PGK terminator from the lower bands of lanes 6 and 7
 * DNA ligation

26th October

 * Incubate for 20mins at 80 degrees celcius
 * Let sit for 5 min.
 * 5uL of above mixture to competent cells
 * 30 min. on ice
 * Heat shock 30s (42 degrees)
 * Add SOC Media (200uL)
 * Incubate 60 min.(37 degrees)
 * Plate 200uL
 * Incubate 37 degrees celcius overnight

27th October
The transformation failed; try again with the same protocol:
 * Using extracts 3 and 7 (+Ligation buffers, Ligase, Plasmid, and Water)


 * Failed again.