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Line 19: |
Line 19: |
| | | |
| | | |
- | Homemade Miniprep | + | '''Homemade Miniprep''' |
| + | |
| 5 samples | | 5 samples |
| + | |
| "Dirty" Mini Preps | | "Dirty" Mini Preps |
- | p1: 50mM Tris-HCl, pH8.0; P2: 200mM NaOH, 1% SDS; P3: 3.0M KAc, pH5.5
| |
| | | |
- | 1. Spin down 1.5 ml cells at 14000 rpm for 2 minutes | + | P1 solution: |
- | Discard supernatant. | + | 50mM Tris-HCl, pH8.0 |
| + | 10mM EDTA, pH8.0 |
| + | |
| + | P2 solution: |
| + | 200mM NaOH, |
| + | 1% SDS; |
| + | |
| + | P3 solution: |
| + | 3.0 M KAc, pH5.5 |
| + | |
| + | |
| + | 1. Spin down 1.5 ml cells at 14000 rpm for 2 minutes. Discard supernatant. |
| + | |
| 2. Resuspend in 300 ul Buffer P1 (with RNAse A) | | 2. Resuspend in 300 ul Buffer P1 (with RNAse A) |
| + | |
| 3. Add 300 ul P2. Mix by inverting. Incubate at room temperature for 5 minutes | | 3. Add 300 ul P2. Mix by inverting. Incubate at room temperature for 5 minutes |
- | 4. Add 300 ul of cold Buffer P3. | + | |
- | Mix by inverting, incubate on ice for 5 minutes | + | 4. Add 300 ul of cold Buffer P3. Mix by inverting, incubate on ice for 5 minutes |
| + | |
| 5. Spin 14000rpm, 10 minutes | | 5. Spin 14000rpm, 10 minutes |
- | 6. Transfer supernatant to a new tube. | + | |
- | Extract with 500 ul Phenol:Chloroform:Isoamyl. | + | 6. Transfer supernatant to a new tube. Extract with 500 ul Phenol:Chloroform:Isoamyl. Spin 14000rpm for 5 minutes |
- | Spin 14000rpm for 5 minutes | + | |
| 7. Transfer 800 ul of the aqueous phase to a new tube | | 7. Transfer 800 ul of the aqueous phase to a new tube |
- | 8. Add 0.7 volumes of isopropanol (560 ul) | + | |
- | Mix by inverting | + | 8. Add 0.7 volumes of isopropanol (560 ul).Mix by inverting. Spin 14000rpm for 5 minutes at 4C |
- | Spin 14000rpm for 5 minutes at 4C | + | |
- | 9. Remove isopropanol. Wash with EtOH 70%. Spin for 2-5 minutes at 14000rpm at 4C. | + | 9. Remove isopropanol. Wash with EtOH 70%. Spin for 2-5 minutes at 14000rpm at 4C.Remove all EtOH. |
- | Remove all EtOH. | + | |
| 10. Air dry. Resuspend in 20 ul TE Buffer. | | 10. Air dry. Resuspend in 20 ul TE Buffer. |
| | | |
- | Electrophoresis: Miniprep of plasmid +Rh1ab on a 1% agarose with 1x TBE | + | '''Electrophoresis: Miniprep of plasmid +Rh1ab on a 1% agarose with 1x TBE''' |
| | | |
| *Photo | | *Photo |
October 18
Inoculation
We inoculated 5 colonies in liquid LB + amp medium
-3 ml medium + 1 colony
We incubated them overnight at 37C with shaking
Prepare other aliquot of primer for the amplification of the gene Rh1ab
Label the tubes well
Homemade Miniprep
5 samples
"Dirty" Mini Preps
P1 solution:
50mM Tris-HCl, pH8.0
10mM EDTA, pH8.0
P2 solution:
200mM NaOH,
1% SDS;
P3 solution:
3.0 M KAc, pH5.5
1. Spin down 1.5 ml cells at 14000 rpm for 2 minutes. Discard supernatant.
2. Resuspend in 300 ul Buffer P1 (with RNAse A)
3. Add 300 ul P2. Mix by inverting. Incubate at room temperature for 5 minutes
4. Add 300 ul of cold Buffer P3. Mix by inverting, incubate on ice for 5 minutes
5. Spin 14000rpm, 10 minutes
6. Transfer supernatant to a new tube. Extract with 500 ul Phenol:Chloroform:Isoamyl. Spin 14000rpm for 5 minutes
7. Transfer 800 ul of the aqueous phase to a new tube
8. Add 0.7 volumes of isopropanol (560 ul).Mix by inverting. Spin 14000rpm for 5 minutes at 4C
9. Remove isopropanol. Wash with EtOH 70%. Spin for 2-5 minutes at 14000rpm at 4C.Remove all EtOH.
10. Air dry. Resuspend in 20 ul TE Buffer.
Electrophoresis: Miniprep of plasmid +Rh1ab on a 1% agarose with 1x TBE
Concentration of sample 5, according to Qubit fluorometer = 128ng/ul
Possible cause of the bands being so faint: We didn't let the pellet resuspend for an hour in step 10
May
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_May_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_May_2010&action=edit 2]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/3_May_2010&action=edit 3]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_May_2010&action=edit 4]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/5_May_2010&action=edit 5]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/6_May_2010&action=edit 6]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/7_May_2010&action=edit 7]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/8_May_2010&action=edit 8]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/9_May_2010&action=edit 9]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/10_May_2010&action=edit 10]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/11_May_2010&action=edit 11]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/12_May_2010&action=edit 12]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/13_May_2010&action=edit 13]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/14_May_2010&action=edit 14]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/15_May_2010&action=edit 15]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/16_May_2010&action=edit 16]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/17_May_2010&action=edit 17]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/18_May_2010&action=edit 18]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/19_May_2010&action=edit 19]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/20_May_2010&action=edit 20]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/21_May_2010&action=edit 21]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/22_May_2010&action=edit 22]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/24_May_2010&action=edit 24]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/25_May_2010&action=edit 25]
| [http://2010.igem.org/Team:Panama/26_May_2010 26]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/27_May_2010&action=edit 27]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/28_May_2010&action=edit 28]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/29_May_2010&action=edit 29]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/30_May_2010&action=edit 30]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/31_May_2010&action=edit 31]
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June
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_June_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_June_2010&action=edit 2]
| [http://2010.igem.org/Team:Panama/3_June_2010 3]
| [http://2010.igem.org/Team:Panama/4_June_2010 4]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/5_June_2010&action=edit 5]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/6_June_2010&action=edit 6]
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[http://2010.igem.org/Team:Panama/7_June_2010 7]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/8_June_2010&action=edit 8]
| [http://2010.igem.org/Team:Panama/9_June_2010 9]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/10_June_2010&action=edit 10]
| [http://2010.igem.org/Team:Panama/11_June_2010 11]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/12_June_2010&action=edit 12]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/13_June_2010&action=edit 13]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/14_June_2010&action=edit 14]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/15_June_2010&action=edit 15]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/16_June_2010&action=edit 16]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/17_June_2010&action=edit 17]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/18_June_2010&action=edit 18]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/19_June_2010&action=edit 19]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/21_June_2010&action=edit 21]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/23_June_2010&action=edit 23]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/24_June_2010&action=edit 24]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/25_June_2010&action=edit 25]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/28_June_2010&action=edit 28]
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July
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_July_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_July_2010&action=edit 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_July_2010&action=edit 3]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_July_2010&action=edit 4]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/5_July_2010&action=edit 5]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/6_July_2010&action=edit 6]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/7_July_2010&action=edit 7]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/8_July_2010&action=edit 8]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/9_July_2010&action=edit 9]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/10_July_2010&action=edit 10]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/11_July_2010&action=edit 11]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/12_July_2010&action=edit 12]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/13_July_2010&action=edit 13]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/14_July_2010&action=edit 14]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/15_July_2010&action=edit 15]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/16_July_2010&action=edit 16]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/17_July_2010&action=edit 17]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/18_July_2010&action=edit 18]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/19_July_2010&action=edit 19]
| [http://2010.igem.org/Team:Panama/20_July_2010 20]
| [http://2010.igem.org/Team:Panama/21_July_2010 21]
| [http://2010.igem.org/Team:Panama/22_July_2010 22]
| [http://2010.igem.org/Team:Panama/23_July_2010 23]
| [http://2010.igem.org/Team:Panama/24_July_2010 24]
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[http://2010.igem.org/Team:Panama/26_July_2010 26]
| [http://2010.igem.org/Team:Panama/27_July_2010 27]
| [http://2010.igem.org/Team:Panama/28_July_2010 28]
| [http://2010.igem.org/Team:Panama/29_July_2010 29]
| [http://2010.igem.org/Team:Panama/30_July_2010 30]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/31_July_2010&action=edit 31]
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August
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_August_2010&action=edit 1]
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[http://2010.igem.org/Team:Panama/2_August_2010 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_August_2010&action=edit 3]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_August_2010&action=edit 4]
| [http://2010.igem.org/Team:Panama/5_August_2010 5]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/6_August_2010&action=edit 6]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/7_August_2010&action=edit 7]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/8_August_2010&action=edit 8]
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[http://2010.igem.org/Team:Panama/9_August_2010 9]
| [http://2010.igem.org/Team:Panama/10_August_2010 10]
| [http://2010.igem.org/Team:Panama/11_August_2010 11]
| [http://2010.igem.org/Team:Panama/12_August_2010 12]
| [http://2010.igem.org/Team:Panama/13_August_2010 13]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/15_August_2010&action=edit 15]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/16_August_2010&action=edit 16]
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[http://2010.igem.org/Team:Panama/23_August_2010 23]
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/28_August_2010&action=edit 28]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/30_August_2010&action=edit 30]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/31_August_2010&action=edit 31]
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September
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_September_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_September_2010&action=edit 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_September_2010&action=edit 3]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/4_September_2010&action=edit 4]
| [http://2010.igem.org/Team:Panama/5_September_2010 5]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/6_September_2010&action=edit 6]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/7_September_2010&action=edit 7]
| [http://2010.igem.org/Team:Panama/8_September_2010 8]
| [http://2010.igem.org/Team:Panama/9_September_2010 9]
| [http://2010.igem.org/Team:Panama/10_September_2010 10]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/11_September_2010&action=edit 11]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/12_September_2010&action=edit 12]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/13_September_2010&action=edit 13]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/14_September_2010&action=edit 14]
| [http://2010.igem.org/Team:Panama/15_September_2010 15]
| [http://2010.igem.org/Team:Panama/16_September_2010 16]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/17_September_2010&action=edit 17]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/18_September_2010&action=edit 18]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/19_September_2010&action=edit 19]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/20_September_2010&action=edit 20]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/21_September_2010&action=edit 21]
| [http://2010.igem.org/Team:Panama/22_September_2010 22]
| [http://2010.igem.org/Team:Panama/23_September_2010 23]
| [http://2010.igem.org/Team:Panama/24_September_2010 24]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/25_September_2010&action=edit 25]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/26_September_2010&action=edit 26]
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[http://2010.igem.org/wiki/index.php?title=Team:Panama/27_September_2010&action=edit 27]
| [http://2010.igem.org/Team:Panama/28_September_2010 28]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/29_September_2010&action=edit 29]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/30_September_2010&action=edit 30]
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October
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/1_October_2010&action=edit 1]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/2_October_2010&action=edit 2]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_October_2010&action=edit 3]
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[http://2010.igem.org/Team:Panama/4_October_2010 4]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/5_October_2010&action=edit 5]
| [http://2010.igem.org/Team:Panama/6_October_2010 6]
| [http://2010.igem.org/Team:Panama/7_October_2010 7]
| [http://2010.igem.org/Team:Panama/8_October_2010 8]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/9_October_2010&action=edit 9]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/10_October_2010&action=edit 10]
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[http://2010.igem.org/Team:Panama/11_October_2010 11]
| [http://2010.igem.org/Team:Panama/12_October_2010 12]
| [http://2010.igem.org/Team:Panama/13_October_2010 13]
| [http://2010.igem.org/Team:Panama/14_October_2010 14]
| [http://2010.igem.org/Team:Panama/15_October_2010 15]
| [http://2010.igem.org/Team:Panama/16_October_2010 16]
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[http://2010.igem.org/Team:Panama/18_October_2010 18]
| [http://2010.igem.org/Team:Panama/19_October_2010 19]
| [http://2010.igem.org/Team:Panama/20_October_2010 20]
| [http://2010.igem.org/Team:Panama/21_October_2010 21]
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| [http://2010.igem.org/Team:Panama/23_October_2010 23]
| [http://2010.igem.org/Team:Panama/24_October_2010 24]
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[http://2010.igem.org/Team:Panama/25_October_2010 25]
| [http://2010.igem.org/Team:Panama/26_October_2010 26]
| [http://2010.igem.org/Team:Panama/27_October_2010 27]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/28_October_2010&action=edit 28]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/29_October_2010&action=edit 29]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/30_October_2010&action=edit 30]
| [http://2010.igem.org/wiki/index.php?title=Team:Panama/31_October_2010&action=edit 31]
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