Team:Illinois/Antibody GPCR Fusion Notebook

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== 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)


== 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
** Result: Failed
* Obtained more yeast from Dr. Zhao


== 25th August ==
* Prepared overnight culture for DNA extraction (3:27pm)


== 26th August ==
* Attempted DNA extraction
* Prepped overnight culture


== 27th August ==
* Performed PCR: PGK Terminator

{| class="wikitable" border="1"
|-
|Buffer G
|12.5uL
|x4
|50uL
|-
|Forward Primer
|0.5uL
|x4
|2uL
|-
|Reverse Primer
|0.5uL
|x4
|2uL
|-
|H2O
|10.8uL
|x4
|43.2uL
|-
|Taq
|0.2uL
|x4
|0.8uL
|-
|template
|colspan="4"|0.5ul 
|-
|Negative control
|3 H2O
|}


*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

{| class="wikitable" border="1"
|-
|Mastermix
|10uL
|x3
|30uL
|-
|Forward Primer
|0.5uL
|x3
|1.5uL
|-
|Reverse Primer
|0.5uL
|x3
|1.5uL
|-
|Template
|10uL
|x3
|30uL
|-
|H2O
|10.8uL
|x4
|43.2uL
|-
|Negative control
|25uL H2O
|}

== 3rd September ==
* Ran gel 
** Ladder lane 7 
** Sample 7 spilled
** 1% agarose 
** 120V 
*** Too low
** 50 minutes

== 8th September ==
* PCR: PGK Terminator

{| class="wikitable" border="1"
|-
|Mastermix
|10uL
|x3
|30uL
|-
|Forward Primer
|0.5uL
|x3
|1.5uL
|-
|Reverse Primer
|0.5uL
|x3
|1.5uL
|-
|Template
|14uL
|x3
|42uL
|-
|}
* 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
** 150V, 50 minutes
** No sign of DNA
* Ladder from Courtney


== 10th September ==
* Split culture
* Ran gel from 8th September again
** 150V, 50 minutes
** 0.75% gel
** Ladder from Courtney

== 11th September ==
* PCR: PGK Terminator

{| class="wikitable" border="1"
|-
|Mastermix
|10uL
|x3
|30uL
|-
|Forward Primer
|0.5uL
|x3
|1.5uL
|-
|Reverse Primer
|0.5uL
|x3
|1.5uL
|-
|Template
|14uL
|x3
|42uL
|-
|}

== 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)
 
{| class="wikitable" border="1"
|-
|5x Phusion HF Buffer
|10uL
|x3
|30uL
|-
|10mM dNTPs
|1uL
|x3
|3uL
|-
|Primer A(Forward)
|1uL
|x3
|3uL
|-
|Primer B(Reverse)
|1uL
|x3
|3uL
|-
|Template(1)
|10uL
|x3
|30uL
|-
|Phusions DNA polymerase
|0.5uL
|x3
|1.5uL 
|-
|H2O
|26.5uL
|x3
|79.5uL
|}

== 18th September ==
* Ran reaction mentioned on 15th September
* Extracted DNA from gel from 8th September (PGK Terminator)

{| class="wikitable" border="1"
|-
|Tube
|Gel(g)
|-
|1
|0.332
|-
|2
|0.278
|-
|3
|0.307
|-
|4
|0.349
|-
|5
|0.385
|}

== 19th September == 
* Gel of PGK Promotor has no DNA present


== 23rd September ==
* PCR: Fus1 Downstream
* EPICENTRE Bioetechnologies - MasterAmp(TM) Taq DNA Polymerase

* Per 50uL of reaction,

{| class="wikitable" border="1"
|-
|MasterAmp Taq 10x PCR Buffer
|5uL
|x3
|15uL
|-
|1mM dNTPs
|1uL
|x3
|3uL
|-
|Primer 1
|1uL
|x3
|3uL
|-
|Primer 2
|1uL
|x3
|3uL
|-
|25mM MgCl2
|6.75uL
|x3
|20.25uL
|-
|MasterAmp 10x PCR Enhancer
|0 or 15uL
|x3
|45uL 
|-
|Taq DNA Polymerase
|0.25uL
|x3
|0.75uL
|-
|template 2
|20uL
|x3
|60uL
|}

* PCR Settings:
** 4mins, 94 degree celcius
** 30s, 94 degree celcius
** 30s, 56 degree celcius
** 1 min, 72 degree celcius -- to step 2 -- 30x
** 7 min, 72 degree celcius

== 24th September == 
* Ran gel of Fus1 Downstream
** Result: No DNA present on gel


== 25th September ==
* PCR: Fus1 Upstream
*same settings as September 23rd
*Template 3


== 30th September ==
* PCR: Fus1 Upstream
*same settings as September 23rd
*Template 4 and 1a

== 1st October ==
* PCR: Ste2


== 8th October ==
* PCR: PGK Terminator
** Use DNA extracted from gel on 18th September
* Also extracted DNA from gel from 30th September


== 9th October ==
* PCR: Ste2
* Template used is product from 1st October
* FIll IN TABLE


* PCR: Fus1 Upstream
* Template used was extracted from 30th September on 8th September
* Fill in PCR TABLE


* Ran Ste2 gel


== 14th October ==
* Fus1 PCR 
* Template is reaction from 12th October

== 15th October ==
* Gel of Fus1 -> 3 streaks -> no ladder




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