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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
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| [http://2010.igem.org/wiki/index.php?title=Team:Panama/3_September_2010&action=edit 3]
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