"

From 2012.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

==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 [[File:PCR Purified eGFP &amp; plsr.JPG|thumb|120px|alt=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 [[File:pET-26b-luxSnocut071712.JPG|thumb|120px|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.

Return to Team:Penn/Notebook/Biofilms.