Team:Charlottesville RS/Notebook/material

From 2014hs.igem.org

(Difference between revisions)
(Extraction of RFP and Iso)
(Methods)
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Before lab, bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. Pelleted cells using tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. Supernatant was poured off into liquid waste container. Tapped the tube to loosen cell pellet, and pipetted 5 mL of cold CaCL2 solution. Incubated tube on ice for 20 minutes. After incubation, spun cells for 5 minutes at 5000 x g (2500 rpm). Poured off supernatant into liquid waste container. Used pipette to add 1 mL of cold CaCl2 solution and resuspended cells.
Before lab, bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. Pelleted cells using tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. Supernatant was poured off into liquid waste container. Tapped the tube to loosen cell pellet, and pipetted 5 mL of cold CaCL2 solution. Incubated tube on ice for 20 minutes. After incubation, spun cells for 5 minutes at 5000 x g (2500 rpm). Poured off supernatant into liquid waste container. Used pipette to add 1 mL of cold CaCl2 solution and resuspended cells.
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===Rehydration of RFP and Iso===
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===Rehydration of RFP and Alcohol Acetyltransferase I===
Pipetted 10uL of dH2O into the well of corresponding Biobrick-standard part. Pipetted up and down, then let sit for 5 minutes. Tranformed 5 uL of the re-suspended DNA into desired cells, plate, and grew overnight.
Pipetted 10uL of dH2O into the well of corresponding Biobrick-standard part. Pipetted up and down, then let sit for 5 minutes. Tranformed 5 uL of the re-suspended DNA into desired cells, plate, and grew overnight.
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===Transformation of RFP and Iso===
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===Transformation of RFP and Alcohol Acetyltransferase I===
Labeled tubes as ''Transformation Control'','' Ligation: New Part'', and ''Ligation Control''. Added 5uL of RFP Control DNA into ''Transformation Control''. Added 2uL of New Part ligation product into ''Ligation: New Part''. Added 2uL of the RFP Control ligation product into the ''Ligation Control''. Put tubes on ice to pre-chill, and thawed one competent cell tube on ice. Pipetted 50uL of competent cells into each 2.0mL micro-centrifuge tube. Incubated DNA and cell mixtures on ice for 30 minutes. During incubation, preheated waterbath to 42˚C. Placed tubes into waterbath for 60 seconds, then incubated on ice for 5 minutes. Added 200uL of SOC media to each tube. Incubated tubes at 37˚C for 2 hours.  Pipetted 200uL of each tube onto respective plates. Spread over surface using glass beads. Incubated plates facing down for 37˚C for 12-14 hours.
Labeled tubes as ''Transformation Control'','' Ligation: New Part'', and ''Ligation Control''. Added 5uL of RFP Control DNA into ''Transformation Control''. Added 2uL of New Part ligation product into ''Ligation: New Part''. Added 2uL of the RFP Control ligation product into the ''Ligation Control''. Put tubes on ice to pre-chill, and thawed one competent cell tube on ice. Pipetted 50uL of competent cells into each 2.0mL micro-centrifuge tube. Incubated DNA and cell mixtures on ice for 30 minutes. During incubation, preheated waterbath to 42˚C. Placed tubes into waterbath for 60 seconds, then incubated on ice for 5 minutes. Added 200uL of SOC media to each tube. Incubated tubes at 37˚C for 2 hours.  Pipetted 200uL of each tube onto respective plates. Spread over surface using glass beads. Incubated plates facing down for 37˚C for 12-14 hours.
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Spun down two culture tubes for 3 minutes at 8000 rpm. Poured supernatant into biological waste container. Pipetted 250uL of Buffer P1 into each 14mL culture tube, and resuspended pellet. Labeled 1.7mL micro-centrifuge tubes with respective part names, and transferred resuspended parts in. Pipetted 250uL of Buffer P2 into each micro-centrifuge tube. Flipped tubes to mix solution. Pipetted 350uL of Buffer N3 into each tube. Flipped tubes to mix solution. Spun down tubes in micro-centrifuge for 10 minutes at 13,000 rpm. Labeled spin columns for each tube, and pipetted supernatant to the respective spin columns. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 500uL of Buffer PB into each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 750uL of Buffer PE to each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Spun down spin columns at 13,000 rpm for 1 minute to remove remaining buffer. Labeled sterile 1.7mL micro-centrifuge tubes with part names. Transferred the appropriate filter tube to the respective tubes. Pipetted 50uL of dH2O to each filter tube. Let samples sit for 1 minute, then spun down tubes at 13,000 rpm for 1 minute. Stored micro-centrifuge tubes at 20˚C.
Spun down two culture tubes for 3 minutes at 8000 rpm. Poured supernatant into biological waste container. Pipetted 250uL of Buffer P1 into each 14mL culture tube, and resuspended pellet. Labeled 1.7mL micro-centrifuge tubes with respective part names, and transferred resuspended parts in. Pipetted 250uL of Buffer P2 into each micro-centrifuge tube. Flipped tubes to mix solution. Pipetted 350uL of Buffer N3 into each tube. Flipped tubes to mix solution. Spun down tubes in micro-centrifuge for 10 minutes at 13,000 rpm. Labeled spin columns for each tube, and pipetted supernatant to the respective spin columns. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 500uL of Buffer PB into each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 750uL of Buffer PE to each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Spun down spin columns at 13,000 rpm for 1 minute to remove remaining buffer. Labeled sterile 1.7mL micro-centrifuge tubes with part names. Transferred the appropriate filter tube to the respective tubes. Pipetted 50uL of dH2O to each filter tube. Let samples sit for 1 minute, then spun down tubes at 13,000 rpm for 1 minute. Stored micro-centrifuge tubes at 20˚C.
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===Digestion of RFP and Iso===
+
===Digestion of RFP and Alcohol Acetyltransferase I===
===Digestion of PSB1C3===
===Digestion of PSB1C3===
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===Ligation of RFP, Iso, and PSB1C3===
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===Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3===
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+
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===Transformation of RFP, Iso, and PSB1C3===
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Revision as of 14:06, 2 May 2014

Material

Buffers & Solution

Kits

Marker

Enzymes

Plasmidvectors

Synthetic oligonucleotides

Phages

Bacteria

Bacteria Growth Media

Methods

Preparing competent cells

Before lab, bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. Pelleted cells using tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. Supernatant was poured off into liquid waste container. Tapped the tube to loosen cell pellet, and pipetted 5 mL of cold CaCL2 solution. Incubated tube on ice for 20 minutes. After incubation, spun cells for 5 minutes at 5000 x g (2500 rpm). Poured off supernatant into liquid waste container. Used pipette to add 1 mL of cold CaCl2 solution and resuspended cells.

Rehydration of RFP and Alcohol Acetyltransferase I

Pipetted 10uL of dH2O into the well of corresponding Biobrick-standard part. Pipetted up and down, then let sit for 5 minutes. Tranformed 5 uL of the re-suspended DNA into desired cells, plate, and grew overnight.

Transformation of RFP and Alcohol Acetyltransferase I

Labeled tubes as Transformation Control, Ligation: New Part, and Ligation Control. Added 5uL of RFP Control DNA into Transformation Control. Added 2uL of New Part ligation product into Ligation: New Part. Added 2uL of the RFP Control ligation product into the Ligation Control. Put tubes on ice to pre-chill, and thawed one competent cell tube on ice. Pipetted 50uL of competent cells into each 2.0mL micro-centrifuge tube. Incubated DNA and cell mixtures on ice for 30 minutes. During incubation, preheated waterbath to 42˚C. Placed tubes into waterbath for 60 seconds, then incubated on ice for 5 minutes. Added 200uL of SOC media to each tube. Incubated tubes at 37˚C for 2 hours. Pipetted 200uL of each tube onto respective plates. Spread over surface using glass beads. Incubated plates facing down for 37˚C for 12-14 hours.

Extraction of RFP and Alcohol Acetyltransferase I

Spun down two culture tubes for 3 minutes at 8000 rpm. Poured supernatant into biological waste container. Pipetted 250uL of Buffer P1 into each 14mL culture tube, and resuspended pellet. Labeled 1.7mL micro-centrifuge tubes with respective part names, and transferred resuspended parts in. Pipetted 250uL of Buffer P2 into each micro-centrifuge tube. Flipped tubes to mix solution. Pipetted 350uL of Buffer N3 into each tube. Flipped tubes to mix solution. Spun down tubes in micro-centrifuge for 10 minutes at 13,000 rpm. Labeled spin columns for each tube, and pipetted supernatant to the respective spin columns. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 500uL of Buffer PB into each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Pipetted 750uL of Buffer PE to each spin column. Spun down spin columns at 13,000 rpm for 1 minute. Poured flow through into chemical waste beaker. Spun down spin columns at 13,000 rpm for 1 minute to remove remaining buffer. Labeled sterile 1.7mL micro-centrifuge tubes with part names. Transferred the appropriate filter tube to the respective tubes. Pipetted 50uL of dH2O to each filter tube. Let samples sit for 1 minute, then spun down tubes at 13,000 rpm for 1 minute. Stored micro-centrifuge tubes at 20˚C.

Digestion of RFP and Alcohol Acetyltransferase I

Digestion of PSB1C3

Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3