Team:Penn/Notebook/Biofilms

From 2012.igem.org

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**Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
**Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
*Nikita, Ashwin, Avin all have acceptable sterile practice
*Nikita, Ashwin, Avin all have acceptable sterile practice
 +
*Resuspended IDT luxS synthesized DNA
 +
*Transformed synthesized luxS into DH5a
==7/11/12==
==7/11/12==
*Ran 2% gel of digest, did not see signs of any restriction
*Ran 2% gel of digest, did not see signs of any restriction
*Re-planned pET-26b digest troubleshooting
*Re-planned pET-26b digest troubleshooting
-
*Resuspended IDT luxS synthesized DNA
+
*Selected luxS colonies
-
*Transformed synthesized luxS into DH5a
+
 
-
*Thawed out and cultured
+
==7/12/12==
 +
*Thawed out 293T-CMV-Gaussia cells from Sarkar LN2
 +
*Miniprepped luxS colony, digested w/ BamHI-HF & EcoRI-HF
 +
*Performed gel extraction of luxS fragment
 +
 
 +
==7/13/12==
 +
*Passaged 293T-CMV-Gaussia cells 1:10 & 1:5 for weekend
 +
*Ligation of luxS w/ pET-26b
 +
*Ligation rxn transformed into DH5a
 +
 
 +
==7/14/12==
 +
*Ligation showed successful transformants
 +
 
 +
==7/15/12==
 +
*Colonies selected by Mike, grown in 10mL LB
 +
 
 +
==7/16/12==
 +
*SAAST Presentation
 +
*Miniprep of 10mL pET-26b-luxS cultures
 +
*Ran gel of minipreps from 3 pET-26b-luxS colonies to confirm successful ligation
 +
*pET-26b-luxS #1/#2 are slightly larger, indicating successful ligation, not as sure for pET-26b-luxS #3
 +
*Last lane is unligated pET-26b vector
 +
[[File:pET-26b-luxSnocut071712.JPG|thumb|120px|northeast|alt=SCIENCE.|Image of 1% Agarose Gel of Miniprep products of pET-26b-luxS from three colonies]]
 +
 
 +
==7/17/12==
 +
*Ran gel to determine if insertion of pET-26b-luxS was correctly ligated
 +
**Gel showed there was a possible size shift corresponding to insertion in all three selected colonies of pET-26b-luxS colonies
 +
[[File:PCRoptimization071812.JPG|thumb|120px|right|alt=SCIENCE.|Image of 2% Agarose Gel of PCR products of eGFP (.85kb) & plsr (.1kb)]]
 +
**From left to right (optimum temperatures bolded): Ladder, plsr @45C,<b>52C</b>,58C,<b>65C</b>, egfp @52C,<b>58C</b>,65C
 +
*Performed 100uL PCR of plsr @65 & 100uL PCR of egfp @58 for further ligation experiments.

Latest revision as of 19:00, 18 July 2012

Contents

6/6/12

  • Set up equipment
  • Autoclaved 2x 1L Sterile ddH2O
  • Autoclaved 500 mL LB Broth for scale up of luxS (Plate 3 Well 2H, pSB1A2), plsr (Plate 2 Well 14H in pSB1A2 resistance to Amp)
  • Added iGEM logo to wiki

6/7/12

  • Resuspended available biobricks luxS & plsr (resuspended in 10uL ddH2O)
  • Resuspended DNA transformed into DH5α (40uL DH5α+2uL DNA) & plated on LB plates w/ 15 uL of 100 mg/mL Amp spread on surface
  • Requested lsrR & lsrK from iGEM HQ

6/8/12

  • iGEM Wiki now split into two separate Notebooks
  • Plenty of colonies, too many to count(TMTC) grew from plsr & luxS transformation from 6/7/12
    • Selected one colony each
    • Grew in 10 mL LB+Amp (100 ug/mL)
  • Contacted researchers for V. harveyi bioassay & lysostaphin, V. harveyi must be purchased through ATCC ($300), lysostaphin obtained thru MTA.

6/9/12

  • ~2mL of LB evaporated during incubation
  • Cells spun down @ 5000 rpm for 10 min
  • Miniprepped 4x Lysis into 1 column
  • [plsr]=112 ng/uL
  • [luxS]=114 ng/uL

6/11/12-6/16/12

  • Dry lab work

6/18/12

  • Obtained & resuspended primers for eGFP & plsr

6/19/12

  • performed PCR w/ NEB standard taq kit w/ primers for eGFP & plsr
  • Tested 2 annealing temperatures, 55C and 50C
  • Ran gel w/ PCR product (2% agarose), saw clear eGFP bands, but possible .1kb obscured by loading dye.

6/20/12

  • Performed qiagen PCR purification & nanodropped (it like it's hot)

6/21/12

  • Ran gel of yesterday's PCR purification
SCIENCE.
Image of 2% Agarose Gel of post-purification PCR products of eGFP (.85kb) & plsr (.1kb)
    • EtBr Gel 2% Agarose
      • Lane 1: NEB 2-log ladder (1 ug loaded)
      • Lane 2: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 55C annealing temp.
      • Lane 3: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 55C annealing temp.
      • Lane 4: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 50C annealing temp.
      • Lane 5: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 50C annealing temp.
      • Lane6/7: Null
      • Lane 8: 1kb ladder left at rt o/n
  • Obtained & transformed pET-26b

6/22/12

  • Miniprepped pet26b and digested with BglII and EcorR

6/25/12

  • Ran gel of eGFP/plsr ligation
  • Ordered restriction enzymes from the Cell Center

6/26/12

  • Attempted Gel Purification of digest products from 2/25/12
    • Yields for Gel purification were too low to be useful (>1 ng/uL)
  • Digested purified PCR rxns (annealing temperature=55C)

6/27/12

Ran gel of digested PCR rxns (55C), found no evidence of successful digestion.

6/28/12

Purified digest and attempted

6/29/12

  • Performed digest of pET-26b, eGFP, and plsr
  • Spread Biobrick shipment on Amp plates (lsrR, lsrK)

7/2/12

  • Selected colonies from lsrK and lsrR streaked plates
    • Grew in 10mL TB Broth

7/3/12

  • Ran gel of ligated product, no size shift, unlikely that ligation was successful.
  • Ligation rxn transformed to make sure

7/5/12

  • Ligation transformation of pET-26-plsr-GFP failed, no colonies present
  • Talked to Dan:
    • When using primers to introduce restriction sites into


7/6/12

  • Redesigned primers for plsr & egfp
  • Set up TC Incubator & hood
  • Contacted people in the FDA thru personal contacts for human practices

7/9/12

  • Made Inactivating Solution for restriction digest
    • 50mM EDTA (pH=8.0)
    • 50% Glycerol
  • Planned test restriction digest pET-26b troubleshooting tomorrow
  • Trained new users for Tissue Culture

7/10/12

  • Troubleshooting of pET-26 digestion
    • Digest of pET-26b w/ BglII and XhoI
    • Time course:
    • 10 min
    • 30 min
    • 3 hr
    • 6 hr
    • o/n
    • Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
  • Nikita, Ashwin, Avin all have acceptable sterile practice
  • Resuspended IDT luxS synthesized DNA
  • Transformed synthesized luxS into DH5a

7/11/12

  • Ran 2% gel of digest, did not see signs of any restriction
  • Re-planned pET-26b digest troubleshooting
  • Selected luxS colonies

7/12/12

  • Thawed out 293T-CMV-Gaussia cells from Sarkar LN2
  • Miniprepped luxS colony, digested w/ BamHI-HF & EcoRI-HF
  • Performed gel extraction of luxS fragment

7/13/12

  • Passaged 293T-CMV-Gaussia cells 1:10 & 1:5 for weekend
  • Ligation of luxS w/ pET-26b
  • Ligation rxn transformed into DH5a

7/14/12

  • Ligation showed successful transformants

7/15/12

  • Colonies selected by Mike, grown in 10mL LB

7/16/12

  • SAAST Presentation
  • Miniprep of 10mL pET-26b-luxS cultures
  • Ran gel of minipreps from 3 pET-26b-luxS colonies to confirm successful ligation
  • pET-26b-luxS #1/#2 are slightly larger, indicating successful ligation, not as sure for pET-26b-luxS #3
  • Last lane is unligated pET-26b vector
SCIENCE.
Image of 1% Agarose Gel of Miniprep products of pET-26b-luxS from three colonies

7/17/12

  • Ran gel to determine if insertion of pET-26b-luxS was correctly ligated
    • Gel showed there was a possible size shift corresponding to insertion in all three selected colonies of pET-26b-luxS colonies
SCIENCE.
Image of 2% Agarose Gel of PCR products of eGFP (.85kb) & plsr (.1kb)
    • From left to right (optimum temperatures bolded): Ladder, plsr @45C,52C,58C,65C, egfp @52C,58C,65C
  • Performed 100uL PCR of plsr @65 & 100uL PCR of egfp @58 for further ligation experiments.
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