Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

From 2008.igem.org

(Difference between revisions)

Revision as of 18:40, 29 October 2008

1st July

TE Buffer Recipe
- Uses
  - TE used to bring up oligos into solution

- People who know how to do this
  - Joleen, Luke

- Concentration
  - 10mM Tris
  - 1mM EDTA
  - pH 8.0

- Method
  - For 500mL volume
    - Add: 0.1861g EDTA; 0.6057g Tris to 1 liter flask
    - Bring it up to 500mL with Deionized water (filtered in ---? container)
    - Put on "corning" mixer; get a larger stir bar from drawer and put on slow ~20 RPM rotation; no heat for 2-3 minutes until fully dissolved.
    - Standardize the pH meter (instructions on sign at prep bench)
    - Put gloves on; put pH electrode in flask but first add stir bar and put on low speed mixing
    - Bring pH to 8.0 (i.e. 8.00 +/- 0.05) by adding base (NaOH) or acid (HCl) in very small drops using a plastic pipette. (Wait for pH meter to eqiullibrate after each drop)

IMP: Put cap back on bottom of pH probe

- - - Put coloured tape along side of flask and label with pH, name and iGEM
    - Cover it with heavy duty aluminium foil (just like a square inch to cover the top) and put a strip of autoclave tape along top of foil
    - Optional: out in big plastic autoclave bin
    - Bring to autoclave room; do not use the big "Beta Star"

    Set on: 15 minutes; ~250 degrees Farenheit = ~121 degree Celcius; "liquid" run; for "operator #" just press enter; then "Run"; tubes = ~ 45 minutes; total to run since it must cool down and decompress

- - - (Some extra side notes still to add)

TAE Buffer Recipe

TBE Buffer Recipe
- - 1 liter of 5x TBE Running Buffer, pH 8.13-8.23

- Materials
  - Tris-base - 54.0g
  - Boric Acid - 27.5g
  - EDTA - 2.92g
  - DI Water - 1.0L

- Method
  - Stirred with stirring rod for 3-5 minutes

Agarose Gel

2nd July

DNA Sequencing - "Core Sequencing " --?
- (https://unicorm.biotec.uiuc.edu) -> "create login account" -> go to "payment manager"
- Primer: One end primer, included in per tube, 10uM concentration of primer
- 30-40 ng/uL? sample concentration
- Protocols/prep online
- ---? to view chromatograms on site
- $5 for sequencing; $2.50 for "--? to load"
- Purify with PAGE before bringing in

1st July

PCR Synthesis of BiFC Genes
- from "PCR-based Synthesis of Long DNA Sequences" 2006 Nature Protocols
- Attempted synthesis of Venus Fluorescent Protein halves (N and C term) fused to HIV gp41 epitope
- Formed gene from ~50nt segment oligos at 30uM
- PCR reactions as described in paper; 50uL reaction volume
- 'FivePrime MasterMix' PCR mix
- PCR protocol as in paper for both N terminal

2nd July

Gel #1: Results from July 1 PCR
- Each lane has 2uL orange/blue loading dye; 1% agarose gel
- Lane 1: Hyperladder II; 2: 8uL C term whole rxn; 3: Cterm w/o leftmost oligo; 4: C w/o rightmost; 5: Just outer oligos 6-9: Same except N terminal fragments
- 60 minutes at 140 V then 10 min in EtBr dye
- No great product

3rd July

Gel #2: again of July 1 PCR
- all lanes have 2uL Loading dye; 1% agarose gel
- Lanes 1+10=ladder; 2+4=8uL whole N term gene; 6+8=8uL C term gene
- again, very fuzzy, impure product

8th July

Agarose Gel Purification of Oligos
- Made 4% Agarose gel with 8g Agarose, 40mL TBE, 160mL H2O
- Made 2% with 4g Agarose
- Microwave for 1:35 for optional warming
- Ran total of 2 hours on 80 Volts -> --? (decent?)

TAZ side project
- Adam Z streaked 4 plates with High Elu, Amp m100, Taz Ecoli
- Check for growth tomorrow

Gel Extraction Protocol
- Use microscope slide to cut gel out
- Visualize on Bio unit with plastic shield from drawer below unit attached to slide out unit
- Amount of gel collected(into 1.5mL tubes)
- Oligo #2: 0.0671g -> 402.6g

  Oligo #3: 0.062g -> 362.0g
  Oligo #4: 0.1062g -> 637.2g
  Oligo #5: 0.0747g -> 448.2g
  Oligo #6: 0.0585g -> 351.0g
  Oligo #7: 0.0601g -> 360.6g
  Oligo #8: 0.0440g -> 264.0g
  Oligo #9: 0.0482g -> 289.2g

9th July

- Cells were found in small colonies -> decided to let them grow for 24 more hours
- Plated 4 more E.coli strains with Xgel (spread)
- Cultured 4 vials of E.coli with X gel
- Check back in 36 hours

9th July

PCR amplification of July 1 PCR
- Hope to get more product, specifically C terminal half
- half of 50uL of pcr reaction volume obtained as 'oligo mix'
- Tube 1: 25 uL oligo mix +1uL of each outer oligo + 20uL Mango MasterMix
- Tube 2: 3uL of oligos 1-5, plus 20uL MasterMix; 50uL total volume with added water
- Tube 3: 3uL oligos 6-10 plus 20uL MasterMix

9th July

Gel #4: Analysis of July 9 PCR
- 1% Agarose gel
- Lane 1: Hyperladder II; 3: 8uL C-term protein (tube1); 5: 8uL tube2; 7: 8uL tube3
- Run 50 minutes at 140 Volts
- Again, lackluster: huge streak seen above and below where we expected product
- Try Again

11th July

Temperature-gradient PCR of Unpurified Oligos
Below is the gel of this reaction at different annealing temps
- 1% agarose gel, run for 50 mins at 140V
- Lane 1: Hyperladder II; 2: 10uL from rxn with 50° annealing temp; 3: 50.9°; 4: 52.8°; 5: 56°; 6L 58.8°; 7: 59.8; Lane 8: 0.6uL 100bp ladder
- Some improved results in mid-range of annealing temp, but clearly have grossly impure product and need to regroup
- Try purifying the 50nt oligos we ordered: likely only ~50% pure from factory

14th July

PAGE Purification of Oligos
- 10% premade PAGE gel w/ 10 50uL wells
- added 40uL of each oligo to well and 8uL loading dye
- Just did this for C terminal protein; also only the inner oligos since the outer are smaller and probably already pure
- Lane 1 and 10 ladder; lanes 2-9 correspond to oligos 2-9 respectively for the C term protein synthesis
- Loaded into PAGE chamber
- Ran for 70 mins at 140V
- Very smeared results-> used too much volume in each well

15th July

Redo of PAGE Purification of Oligos
- Same as July 14th protocol except used 20uL of each oligo instead of 40uL to prevent overflowing
- Also used 10uL and not 8uL loading dye
- Ran for 30 minutes at 200 Volts
- Put in EtBr dye for 15 mins and imaged
- HUGE streaks for each of our products; either gel was run poorly or we have like 10% purity of oligos
- Conclusion: this PCR gene synthesis method will not work
- Review literature for other approaches

20th July

Obtained a Citrine Fluorescent protein from UIUC Rao lab
- On bacterial plasmid
- Will use instead of synthesizing whole gene de novo
- Sequence known so can design overlapping primers from our 'novel' part of the gene
- Instead of fusing HIV gp41 fragment to each FP half, will fuse a poly (6X) His tag
- Model system to evaluate BiFC assay: His-tag fused to each FP half
- Bound by mAb against PolyHist -> complementation (hopefully)

30th July

NEW Gene Synthesis Protocol
- From 'Gene2Oligo' tool available online
- Breaks gene up into ~40nt chunks but OVERLAP FULLY so that even impure oligos can create pure product
- AminoAcid Sequence: overlap CFP from Rao lab-> 20AA flexible peptide linker-> PolyHis Tag-> stop
- This ~100 nt sequence will be synthesized de novo w/ Gene2Oligo Method
- Ordered 6 oligos of ~40nt, brought to 20uM
- PCR as described by Gene2Oligo paper:
- 0.1uM final oligo concentration, plus FivePrime MasterMix, plus 25mM MgCl2, plus water to 50uL
- First performed the Gene2Oligo 'synthesis' step of the protocol

31 July

Gene2Oligo 'Amplification' Step
- As described in paper: 50 uL pcr tube; 20uL MasterMix, 1.5 uL of each 30uM outer primer, 3uL of rxn from July30, plus MgCl2
- Ran w/ PCR reaction from paper
- Product added to gel from other IGEM expt: AWESOME, dark, perfect band right where we expect it
- This method works to synthesize at least smaller (~100nt) segments
- Also, we have a functional collection of the His-Tag, linker, overlap gene
- Next step: amplify out the CFP from the plasmid to fuse to this

5th August

PCR Amplification of Citrine Fluorescent Protein
- Ordered primers brought up w/ water to 300uM
- In each PCR tube: 30uM final concentration of each (forward and reverse) primer, 20uL MasterMix, 1uL out of 50uL plasmid, brought up to 50uL total volume
- PCR protocol: 90° for 50 sec; 50° for 50 sec; 72° for 1:45; repeat this for 30 cycles, then 72° for 5:00, then hold at 4° for ever

6th August

Gel Electrophoresis of 5 August PCR
- 8uL PCR mixture plus 2uL loading dye
- 1.5% agarose gel at 200V for about 50 mins
- EtBr visualization
- Very dim band where we expect product, also a couple other dim bands
- Bad gel? Or bad PCR

6th August

Revised PCR Protocol for CFP Amplification
- Same mixture in tube as August 5th
- BUT: Initial denaturation at 94°C for 2:00
- then: 94° for 1:00, 47° for 1:00, 72° for 1:00; repeated 25 times; then 72° for 7 mins, then hold at 4°

7th August

Gel of August 6 PCR
- 2% Agarose gel, lane 1= 100bp ladder; land 2= 8uL product + 2uL loading dye
- EtBr then imaging, a little darker produce but still nothing great, probably not enough to transfect

3rd September

PCR Amplification of CFP: Round III
- Two reactions using the same protocol as on the 6h of august, but with different PCR Mix:
- 20uL 5Prime MasterMix, OR 25uL MangoMix

7th September

Gel of PCR from September 3
- 1.5% agarose gel, run at 150 Volts for 50 minutes
- Lane 1: loading dye and 5uL 100bp ladder; 2: 8uL MasterMix reaction; 3: 8uL MangoMix rxn
- EtBr visualization
- No great difference between the two pcr mixtures
- Still no great product

8th September

One Last Attempt: PCR Amplification of PCR
- Increased the concentration of primers: 60uM instead of 30uM
- Also, used product from the reaction from September 3 instead of raw plasmid (amplify amplification?)
- Same PCR protocol as on September 3 (except 45° annealing temp)
- Ran 1% agarose gel for 50 mins at 150V
- Lane 1: 5uL 100bp ladder; lane 2: 8uL above PCR product
- Got a decent amount of product, but low purity-> huge smudge
- Impurity possibly due to re-amplification step
- Possible villain: low annealing temps
- Solution: order new amplification oligos with higer temps for annealing

29th September

PCR Amplification of CFP Using Longer Primers
- Longer oligos (~30nt each) used to increase melting temperature; brouth up to 30uM
- PCR mix: 10uL 2X MasterMix (custom made by Matt); 15uL forward primer; 15uL reverse; 10uL PCR product from Sept 8
- Same PCR protocol as from September 3, except annealing temp was set to 52° instead of 45

30th September

Agarose Gel of Sept 29 PCR
- 1% Gel run for 50 mins at 150V
- Lane 1: 5uL Ladder; Lane 2: 8uL Sept 29 reaction product plus loading dye; lane 3 same as lane 2
- Decent amount of product, but a lot of smearing
- Possible cause: we again re-amplified an amplification instead of going from original template

30th September

PCR Amplification of Original CFP Plasmid with Long Primers
- Same program as Sept 29 except set annealing temp to 55°C
- Tube 1: 8uL MasterMix, 15uL forward long primer, 15uL reverse primer, 5uL plasmid, brought up to 50uL total
- Control tube (2) had Mastermix plus control template and primers.

1st October

Agarose Gel of Sept 30 PCR
- 1% Gel run for 50 mins at 150V
- Lane 1: 1kbp ladder; 2: 8uL reaction from sept 30; 3: control reaction from sept 30
- EtBr staining then imaging
- Way too much ladder (use less in future); also very promising but sort of dark band at ~750nt- Exactly where expected!

2nd October

Redo of Oct 1 Gel to Confirm Results
- Lane 1: 2uL 1kBp ladder; 2: 8uL reaction from sept 30; 3: control from Sept 30
- Still too much ladder, but otherwise success! Dark band at 750 again
- Next step-> extract this band

5th October

Repeat of PCR Amplification of Original CFP Plasmid with Long Primers
- Same program and reaction as October 2
- Then do 1% gel at 150V for 50 mins, EtBr and visualize-> same band
- Extract this next

7th October

DNA Extraction from Oct 5 Gel
- 'Invitrogen PureLink Quick Gel Kit'
- Instructions followed directly from kit (see invitrogen website for details)
- ~150mg isolated from VFP gel around band (with 450uL volume used); also ~140mg from control gel (420uL volume)
- Next: attempt to quantify DNA
- [DNA] = 50 ug/ml * Dilution Factor * OD260
- Tube1 VFP amplification DNA: 0.0055ug/uL; Tube 2 control DNA: 0.186 ug/uL
- Nice amount of DNA! Next-> clone this into our vector

22nd October

3rd October

@@ Line 250: / Line 250: @@
 ** Decent amount of product, but a lot of smearing
 ** Possible cause: we again re-amplified an amplification instead of going from original template
 == 30th September ==
@@ Line 271: / Line 270: @@
 ** Next step-> extract this band
-== 3rd October ==
+== 5th October ==
-*
+* Repeat of PCR Amplification of Original CFP Plasmid with Long Primers
-**
+** Same program and reaction as October 2
-**
+** Then do 1% gel at 150V for 50 mins, EtBr and visualize-> same band
-**
+** Extract this next
-**
+== 7th October ==
+* DNA Extraction from Oct 5 Gel
+** 'Invitrogen PureLink Quick Gel Kit'
+** Instructions followed directly from kit (see invitrogen website for details)
+** ~150mg isolated from VFP gel around band (with 450uL volume used); also ~140mg from control gel (420uL volume)
+** Next: attempt to quantify DNA
+** [DNA] = 50 ug/ml * Dilution Factor * OD260
+** Tube1 VFP amplification DNA: 0.0055ug/uL; Tube 2 control DNA: 0.186 ug/uL
+** Nice amount of DNA! Next-> clone this into our vector
-== 3rd October ==
+== 22nd October ==
 *
 **

Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

From 2008.igem.org

Revision as of 18:40, 29 October 2008

Contents

1st July

2nd July

1st July

2nd July

3rd July

8th July

9th July

9th July

9th July

11th July

14th July

15th July

20th July

30th July

31 July

5th August

6th August

6th August

7th August

3rd September

7th September

8th September

29th September

30th September

30th September

1st October

2nd October

5th October

7th October

22nd October

3rd October