- Construction of GFP marker for a part which will be secreted using T3SS
- Ordered primers for construction for same part
Digestion of GFP and Double Terminator
Parts Information
| Description
| BioBrick No.
| Well No.
| Length
| Plasmid
|
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|
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|
|
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|
| GFP
| BBa_E0040
| 1-14K
| 720bp
| pSB1A3
|
| double terminator
| BBa_B0015
| 1-23L
| 129bp
| pSB1AK3
|
| pSB1A3
| pSB1A3
| -
| 2157bp
| pSB1A3
|
1-14K and pSB1A3 had purified with mycrocon, and 1-23L had extracted from a gel before.
- electrophoresed 1ul of 1-14K and 1-23L added 0.5ul of 6×sample buffer to estimate concentration of each solution.
- estimated concentration from photo of electrophoresys. But I forgot to electrophorese pSB1A3 solution with the other samples, so pSB1A3 solution was done by other person.
- made digestion recipes based on each concentrations(below). Why pSB1A3 recipe is two, because I firstly made 30ul of pSB1A3 solution, but I found it was insufficient to ligate parts, so I made more 50ul of it after.
| 1-14K
| 200 ng/ul
|
| 1-23L
| 120 ng/ul
|
| pSB1A3
| 2.5 ng/ul
|
| 1-23L
| 0.5 uL
|
| 10x M buffer
| 5 uL
|
| 0.1%BSA
| 5 uL
|
| Xba I
| 4 uL
|
| Pst I
| 0.5 uL
|
| DW
| 35 uL
|
| Total
| 50 uL
|
| 1-14K
| 1.5 uL
|
| 10x H buffer
| 2 uL
|
| 0.1% BSA
| 2 uL
|
| EcoR I
| 1 uL
|
| Spe I
| 0.5 uL
|
| DW
| 13 uL
|
| Total
| 20 uL
|
| pSB1A3
| 20 uL
|
| 10x H buffer
| 3 uL
|
| 0.1% BSA
| 3 uL
|
| EcoR I
| 0.5 uL
|
| Pst I
| 0.5 uL
|
| DW
| 3 uL
|
| Total
| 30 uL
|
| pSB1A3
| 30 uL
|
| 10x H buffer
| 5 uL
|
| 0.1% BSA
| 5 uL
|
| EcoR I
| 0.5 uL
|
| Pst I
| 0.5 uL
|
| DW
| 9 uL
|
| Total
| 30 uL
|
- put each solutions into 37C incubator.
- 1-23L solution was put about a half and two hours, 1-14K solution was done about a half and an hour, 30ul of pSB1A3 solution was done about an hour, and 50ul of pSB1A3 was done about a half hour.
- electrophoresed each solutions added 6x sample buffer.
- put 12uls each into wells of a gel like below.
| 1
| λ/HindIII, EcoR I
|
| 2~3
| 1-14K
|
| 4~8
| 1-23L
|
| 9~16
| pSB1A3
|
Result
- cannot see 1-23L because of overflowing.
- extracted the other samples from a gel.
- dissolve them with 50 ul of Nuclease free water, and they were stocked to freeze in -20C.
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