Minnesota/5 August 2008

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|'''3. Digest Rxn:''' Run a digest reaction on the plasmid prep products. Follow the chart below:
|'''3. Digest Rxn:''' Run a digest reaction on the plasmid prep products. Follow the chart below:
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer!! BSA !! H20 !! Insert DNA !! RE1 !! RE2 !! Total
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|-
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| L/L PCR || 5.0uL || 05uL || 22.5uL || 20.0uL || 1.0uL, EcoRI || 1.0uL, Pst1 || 50.0uL
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|-
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| TetR/p22mnt PCR || 5.0uL || 0.5uL || 22.5uL || 20.0uL || 1.0uL, EcoRI || 1.0uL, Pst1 || 50.0uL
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|-
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| BV || 5.0uL || 0.5uL || 37.5uL || 5.0uL || 1.0uL, EcoRI || 1.0uL, Pst1 || 50.0uL
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|-
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| Lac/LAMBDA + GFP 1 || 5.0uL || 0.5uL || 12.5uL || 30.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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|-
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| Term || 5.0uL || 0.5uL || 37.5uL || 5.0uL || 1.0uL, Xba1 || 1.0uL, Pst1 || 50.0uL
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|-
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| L/L + GFP 2 || 5.0uL || 0.5uL || 7.5uL || 35.0uL || 1.0uL, Spe1 || 1.0uL, Pst1 || 50.0uL
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|}
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|-
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|'''4. Vector Dephosphorylation of Digest Products:''' Dephosphorylated the BV, Lac/LAMBDA + GFP 1, and Lac/LAMBDA + GFP 2. Did not dephosphorylate the PCR products or the terminator. Used 1uL of antarctic phosphatase and 5.6uL of antarctic phosphatase buffer. 30 mins @ 37C in an incubator, then 15 minutes of heat inactivation.
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|-
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|'''5. Run a gel on Digest Terminator Product:''' Using 1% agarose gel, check for correct amount/type of DNA bands. Only run terminator on gel. Will most likely not visualize band because terminator is approx. 40 bp's long.
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|'''6. Gel purification:''' The terminator band was cut out of the electrophoresis gel and purified out using QIAGEN's QuickSpin gel purification protocol. DNA used in ligation below.
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|-
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|'''7. Ligations:''' Ligated the digested, dephosphorylated, and gel purified products. After following the procedure/table of ligations below, we stored the ligated products @ -20C O/N to then transform tomorrow into competent cells. Folow the ligation table below:
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{|border="1" align="left"
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|-
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!| Parts !! 10x Buffer !! T4 DNA Ligase !! Base Vector !! Insert DNA !! H20 !! Total
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|-
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| BV + L/L PCR || 4.0uL || 4.0uL || 1.0uL || 25.0uL || 6.0uL || 40.0uL
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|-
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| BV + T/P || 4.0uL || 4.0uL || 1.0uL || 25.0uL || 6.0uL || 40.0uL
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|-
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| L/L : GFP 1 + Term || 4.0uL || 4.0uL || 4.0uL || 25.0uL || 3.0uL || 40.0uL
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|-
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| L/L : GFP 2 + Term || 4.0uL || 4.0uL || 4.0uL || 25.0uL || 3.0uL || 40.0uL
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|}
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|-
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|'''8. Sequencing Rxns:''' Send in sequences. Follow the mixture below, which was sent in today:
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{|border="1" align="left"
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|-
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!| Parts !! H20 || DNA || primer
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|-
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| TetR/p22mnt + RFP + Term || --- || 8.8uL || 3.2uL
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|-
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| TetR/p22mnt + RFP + Term || --- || 8.8uL || 3.2uL
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|-
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| L/L + GFP 1A || --- || 8.8uL || 3.2uL
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|-
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| L/L + GFP 1E || --- || 8.8uL || 3.2uL
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|-
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| Tetpro + LAMBDAcI + TermA || --- || 8.8uL || 3.2uL
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|-
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| Tetpro + LAMBDAcI + TermB || --- || 8.8uL || 3.2uL
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|-
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| L/L + GFP 2A || --- || 8.8uL || 3.2uL
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|-
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| L/L + GFP 2B || --- || 8.8uL || 3.2uL
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|}

Latest revision as of 21:03, 6 August 2008

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1. Plasmid Prep: Plasmid prep from the 2mL cultures made from picked colonies of (a) TetR/p22mnt + RFP + Term, (b) Lac/LAMBDA + GFP 1, (c) Lac/LAMBDA + GFP 2, (d) Tetpro + LAMBDAcI + term.
2. Spectrophotometry: Spec'ed the plasmid prep products to check for concentration of DNA.
3. Digest Rxn: Run a digest reaction on the plasmid prep products. Follow the chart below:


Parts 10x Buffer BSA H20 Insert DNA RE1 RE2 Total
L/L PCR 5.0uL 05uL 22.5uL 20.0uL 1.0uL, EcoRI 1.0uL, Pst1 50.0uL
TetR/p22mnt PCR 5.0uL 0.5uL 22.5uL 20.0uL 1.0uL, EcoRI 1.0uL, Pst1 50.0uL
BV 5.0uL 0.5uL 37.5uL 5.0uL 1.0uL, EcoRI 1.0uL, Pst1 50.0uL
Lac/LAMBDA + GFP 1 5.0uL 0.5uL 12.5uL 30.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
Term 5.0uL 0.5uL 37.5uL 5.0uL 1.0uL, Xba1 1.0uL, Pst1 50.0uL
L/L + GFP 2 5.0uL 0.5uL 7.5uL 35.0uL 1.0uL, Spe1 1.0uL, Pst1 50.0uL
4. Vector Dephosphorylation of Digest Products: Dephosphorylated the BV, Lac/LAMBDA + GFP 1, and Lac/LAMBDA + GFP 2. Did not dephosphorylate the PCR products or the terminator. Used 1uL of antarctic phosphatase and 5.6uL of antarctic phosphatase buffer. 30 mins @ 37C in an incubator, then 15 minutes of heat inactivation.
5. Run a gel on Digest Terminator Product: Using 1% agarose gel, check for correct amount/type of DNA bands. Only run terminator on gel. Will most likely not visualize band because terminator is approx. 40 bp's long.
6. Gel purification: The terminator band was cut out of the electrophoresis gel and purified out using QIAGEN's QuickSpin gel purification protocol. DNA used in ligation below.
7. Ligations: Ligated the digested, dephosphorylated, and gel purified products. After following the procedure/table of ligations below, we stored the ligated products @ -20C O/N to then transform tomorrow into competent cells. Folow the ligation table below:
Parts 10x Buffer T4 DNA Ligase Base Vector Insert DNA H20 Total
BV + L/L PCR 4.0uL 4.0uL 1.0uL 25.0uL 6.0uL 40.0uL
BV + T/P 4.0uL 4.0uL 1.0uL 25.0uL 6.0uL 40.0uL
L/L : GFP 1 + Term 4.0uL 4.0uL 4.0uL 25.0uL 3.0uL 40.0uL
L/L : GFP 2 + Term 4.0uL 4.0uL 4.0uL 25.0uL 3.0uL 40.0uL


8. Sequencing Rxns: Send in sequences. Follow the mixture below, which was sent in today:


Parts H20 DNA primer
TetR/p22mnt + RFP + Term --- 8.8uL 3.2uL
TetR/p22mnt + RFP + Term --- 8.8uL 3.2uL
L/L + GFP 1A --- 8.8uL 3.2uL
L/L + GFP 1E --- 8.8uL 3.2uL
Tetpro + LAMBDAcI + TermA --- 8.8uL 3.2uL
Tetpro + LAMBDAcI + TermB --- 8.8uL 3.2uL
L/L + GFP 2A --- 8.8uL 3.2uL
L/L + GFP 2B --- 8.8uL 3.2uL
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