Characterization by Micah Madrid https://padlet.com/micah_madrid/hhi5x3rirnsb FUCK YEAH IGEM 2018 LETS DESTROY THIS SHIT en-us 2018-08-15 17:30:56 UTC 2019-07-09 18:11:23 UTC hello@padlet.com SDS -PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis Notes and protocol (7/15) micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273305335 Mutant/Non-Mutant PETase
The Sequence Manipulation Suite: Protein Molecular Weight
Results for 873 residue sequence starting "ATGAATTTCC".
The protein weighs 72.69 kilodaltons

MHETase
The Sequence Manipulation Suite: Protein Molecular Weight
Results for 1904 residue sequence starting "ATGCAGACCA".
The protein weighs 157.34 kilodaltons

4-40 kDa | Up to 20%
12-45 kDa | 15%
10-70 kDa | 12.5%
15-100 kDa | 10%
50-200 kDa | 8%
>200 kDa | 4-6%

We are using an 8% gel

https://www.cytographica.com/lab/acryl2.html
Link to determine variable percentages for resolving gel

]]> 2018-08-15 17:32:19 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273305335 SDS PAGE MIXTURE and Calculations micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273323287 30% Acrylamide/bis 0.5M 
(0.5 x X%)

Tris-HCl, pH 6.8 (Stacking)

1.5M Tris-HCl, pH 8.8 (resolving) 3.75 ml  

10% SDS 150 μl

diH2O 7.28 ml 
(11.03–(0.5 x X%))

TEMED 7.5 μl 

10% APS 75 μl

Total volume 15ml
]]> 2018-08-15 18:42:29 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273323287 8% Gel -- We need three resolving micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273325655 30% Acrylamide/bis 0.5M 4 ml
h20 7.03 ml
Tris 3.75 ml( pH 8.8)
10% SDS  150 uL

10% APS 75 uL
TEMED 7.5 uL

Approximately 15ml
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf

Resolving to 40% to 30% 
3ul -Pa x 3 = 9mL
1ul -H20x3 = 3mL

Stacking 40% to 30%
.975uL Pa x3= 2.925
.325 uL H2o x 3 = .975uL

]]> 2018-08-15 18:51:54 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273325655 Cell Lysate micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273331220 If you want to make quick extracts from whole cells, I would suggest to take 1ml of culture at OD(600)=2.0 (or more or less depending on the density of your culture), spin down the cells and resuspend the pellet in 50 µl of 1xSDS loading buffer. Boil for 10 min, then spin at max. speed for 10 min. The sample will be viscous, but if you take 8 µl from the very top of the liquid, you should easily be able to pipet it. 

I always use this protocol to look at the total lysate and I never have problems with it. If you stick to these values for the OD and for the volumes, then you should get a nice Coomassie-stain, and Western blot should also work perfectly...
]]> 2018-08-15 19:18:45 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273331220 5% Stacking micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273559763 30% Acrylamide/bis 0.5M 1.3ml
h20 5.5ml
Tris 1ml (use pH 6.8)
10% SDS  80ul

10% APS 80ul
TEMED 8ul]]> 2018-08-16 18:10:42 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273559763 Optical Density micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273785920 .976 Control
.862 Cutinase & Mhetase
.873 PETase
.811 Cutinase]]> 2018-08-17 18:15:05 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273785920 (8/17) Resolving (45 mL) karen_deleon1 https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273825166 30% Acrylamide/bis : 12 ml 
H2O: 21.09 mL
Tris: 11.25 mL
10% SDS: 450 uL

10% APS: 225 uL
TEMED: 22.5 uL

Approximately: 45 mL

Stacking
30% acrylamide/bis: 2.6 mL 
H2O: 11 mL 
Tris: 2 mL 
10% SDS: 160 uL 

10% APS: 160 uL 
TEMED: 16 uL 


]]> 2018-08-17 22:48:33 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273825166 (8/17) Gel karen_deleon1 https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273828532 200 voltage for 30 min.
Well 1: ladder
Well 2: Supernatant cut+mhet
Well 3: Supernatant cut
Well 4: Supernatant PET
Well 5: Supernatant Ctrl
Well 6: Pellet cut+mhet
Well 7: Pellet Cut
Well 8: Pellet PET
Well 9: Pellet Ctrl ]]> 2018-08-18 00:10:19 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/273828532 Running Buffer micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368261 10x
5 ml of 20% SDS
30g Tris
144 g of glycine
995 ml H20

1x
500 ul of 20% SDS or 1 ml of 10% SDS
3 g of Tris
14.4 g of glycine
999ml of H20 or to 1000]]> 2018-08-21 17:08:31 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368261 Sample Buffer Unsure of X micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368295 4 ml of H20 
1.25 ml of 0.5M Tris pH 6.8
2ml of 50% glycerol (or sucrose)
2 ml of 10% SDS
.5 before use mercaptoethanol 
.05g of bromophenol blue]]> 2018-08-21 17:08:39 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368295 Sample Prep 2 for Protein Gel micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368338 1. Centrifuge cells (~5 x 107) for 3 min at 15 ,000 x g and resuspend the pellet in an equal volume of 37°C PBS and centrifuge again. 2. Repeat two more times to remove all interfering material (extracellular proteases and growth media).
3. Add 200 μl of hot (95°C) SDS sample 10% solubilization buffer to the pellet and vortex thoroughly.
5. Cool the sample to 20°C and dilute with ~250 μl 2x SDS-PAGE sample buffer. Incubate for another 20 min at room temperature.
Centrifuge the sample solution at 20°C for 30 min at 14,000 x g and harvest the supernatant.
Perform the protein assay. The protein concentration should be ~5 μg/μl.
]]> 2018-08-21 17:08:44 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274368338 Sample Buffer 2X micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274381695 2 mL Tris (1 M, pH 6.8)
4.6 mL glycerol (50%)
1.6 mL SDS (10%)
0.4 mL bromophenol blue (0.5%) 
(.02 g + 400 ul of H20)


0.4 mL β-mercaptoethanol

]]> 2018-08-21 17:50:48 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274381695 Broad Range BioRad Ladder micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274393322 http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006035.pdf]]> 2018-08-21 18:24:26 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274393322 After it runs ask Kylie how to stain micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274393424 If not I can ask tomorrow / if its complicated ]]> 2018-08-21 18:24:43 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274393424 Loading into Gel micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274398871 Load 10 ul of ladder
Load 10-25 of sample (25)
Run at 200V for 35 minutes

]]> 2018-08-21 18:42:26 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274398871 Protein Extraction micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274428604
  • Centrifuge the cell suspension at 2000G for 5-7 min at 4°C. The cells are collected at the bottom of the tube, discard the supernatant.

  • To the cell pellet, add ice-cold PBS and wash the cells by centrifuging at 2000G for 5-7 min at 4°C.

  • Add pop culture (10% of total volume ) to the cell pellet. Agitate the contents in microfuge tubes for 30 min at 27ºC.

  • Centrifuge the tubes at 16000G for 20 min at 4°C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

    • ]]>     2018-08-21 20:45:16 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274428604     Staining and Destaining micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274445829
    Coomassie® R-250 Staining Protocol 

    1. Prepare the staining solution containing 0.1% Coomassie® R-250 in 40% ethanol, 10% acetic acid. 

    1. After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie® Blue R-250 staining solution. Caution: Use caution while performing the following steps using a microwave oven. Do not overheat the staining solutions. 

    1. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. To prevent hazardous, flammable vapors from forming, do not allow the solution to boil. 

    1. Remove the staining container from the microwave oven and gently shake the gel for 15 minutes at room temperature on an orbital shaker. 

    1. Decant the stain and rinse the gel once with deionized water. 

    1. Prepare a destain solution containing 10% ethanol and 7.5% acetic acid. 

    1. Place one or two stained gels in a staining container containing the 100 ml destain solution. 

    1. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. 

    1. Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved.
    ]]>     2018-08-21 23:12:17 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274445829     (08/22) Protein Gel karen_deleon1 https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274702664 Resolving
    30% Acrylamide/bis 0.5M 4 ml
    h20 7.03 ml
    Tris 3.75 ml
    10% SDS  150 uL

    Add exactly before pouring the gel:
    10% APS 75 uL
    TEMED 7.5 uL

    Note: When you pour the gel, you need 7.5 mL of resolving.

    Stacking 
    30% Acrylamide/bis 0.5M 1.3ml
    h20 5.5ml
    Tris 1ml (use pH 6.8)
    10% SDS  80ul

    Add exactly before pouring the gel:
    10% APS 80ul
    TEMED 8ul

    Note: ]]>     2018-08-22 20:02:37 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/274702664     (09/08) Dilutions for Protein Gel karen_deleon1 https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/279114743 How to dilute 40% Acrylamide/bis to 30%
    1. Use C1V1=C2V2 Formula
    2. 0.4x=0.3(14 *amount needed in mL)
    3. x is the amount of Acrylamide needed
    4.  the rest is D.I Water

    How to dilute 20% SDS to 10% SDS
    1. Half of the amount needed will be SDS and the rest will be D.I water 

    How to make APS
    1. 0.1 g of Ammonium Persulfate
    2. 1000 uL of D.I water




    ]]>     2018-09-08 22:47:29 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/279114743     SAMPLE BUFFER 5X micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/280666716
  • 15ml stock solution of western blot loading buffer. Dilute for use.
  • To prepare base solvent add 3ml 20% SDS
  • add 3.75mL 1M Tris buffer at pH 6.8 in a suitable container.
  • Add 9 mg bromphenol blue
  • 2.4ml B-mercaptoethanol and mix well. (do this in the hood.) borrow from todd
  • Add 4.5mL 100%. glycerol to the solution, mix well.
  • Make up to a final volume of 15ml with dH20 and mix again thoroughly
  • Store at 4’C. Dilute to use.



    • ]]>     2018-09-13 05:35:03 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/280666716     How to make Tris-HCl magaly_aguirresanchez https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289961489 -Dissolve 121.14 g Tris (American Bioanalytical #AB14042) in 800 ml dH2O.

    -Adjust pH to 7.0 with the appropriate volume of concentrated HCl. Bring final volume to 1 liter with deionized water

    -Autoclave and store at room temperature.]]>     2018-10-06 22:32:21 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289961489     Tris-HCl ph 8.8 magaly_aguirresanchez https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289961895 -add 6.04g of Tris
    -add 50mL of DI water

    ]]>     2018-10-06 22:40:06 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289961895     Tris-HCl pH 6.8 magaly_aguirresanchez https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289962024 -add 4.856g of Tris
    -add 40mL of DI water]]>     2018-10-06 22:42:03 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/289962024     Sample Prep from Biochem lab noble_woodward https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/304933576
    The standards and unknown protein samples are treated in a boiling water bath for 5 min in electrophoresis buffer containing 5-10% 2-mercaptoethanol and 1% SDS.  Filter, or microfuge, out any precipitated protein.  An equal volume of 40% sucrose is then added to increase the sample density.  The sucrose solution also should contain 0.02% brom(o)phenol blue as a tracking dye.
    ]]>     2018-11-15 17:49:36 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/304933576     HI CJ GO TO END OF THIS TO FIND WORK micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/318610039 2019-01-09 00:10:48 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/318610039 SDS PAGE Gels 15% micah_madrid https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/318610061 http://www.assay-protocol.com/uploads/Clean_Lilaic/SDS%20PAGE.pdf
    Use this,]]>     2019-01-09 00:11:04 UTC https://padlet.com/micah_madrid/hhi5x3rirnsb/wish/318610061