IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week12/Chemical and Light: Difference between revisions
Meng Xiao He (talk | contribs) |
Meng Xiao He (talk | contribs) |
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Lane 12: control | Lane 12: control | ||
=Ligations/Transformations 09/26= | =Ligations/Transformations 09/26= | ||
RE digests (re)done on appropriate minipreps using Fermentas enzymes and extracted using Clonewll gels. | 25min RE digests (re)done on appropriate minipreps using Fermentas enzymes and extracted using Clonewll gels. | ||
We used the Takara ligation kit to do the following ligations (1:6 vector:insert): | We used the Takara ligation kit to do the following ligations (1:6 vector:insert): |
Revision as of 16:16, 26 September 2008
9/22 RE digests
With NEB enzymes, 60 min (buffer 2+BSA):
- 128#1 XPXmnI
- 128#5 XSXmnI
- 128#9 ESXmnI
The XmnI is to cut the backbone, which has a similar length to the mtrB constructs.
With Fermentas enzymes, 25 min:
- 116 SP
- 117 SP
- 108 SP
- 51 EX
- 63 XP
9/23 ligations
The following ligations were performed using 1:6 molar ratio and the Takara kit at 16°C for 45min:
- 128ES+63EX (also did a 5min, 25°C ligation to test whether the extraction method or the ligation conditions are improving the process)
- 128XP+108SP
- 128XP+116SP
- 128XP+117SP
- 51EX+38ES
- Neg ctrl with water as insert
- 117SP+128XS+[63XP]
- 108SP+128XS+[63XP]
- 116SP+128XS+[63XP]
Note that [63] means P63 was added 20 minutes into the ligation (to reduce uptake only of terminator). Prior to addition of ligase, DNA mix was heated to 65°C for 2 minutes and allowed to cool. For ones with 63 added later, the 63 was heated prior to addition, and more ligation mix was added next to make up the volume.
All transformed into DH5α.
9/24 transformation results
Only the P128ES+63EX ligations gave colonies. Both the long and short ligations gave hundreds of colonies, although the long one had more.
9/24 P128+63EX colony PCR
PCR using BBpfx and mtrB-internal500. Should produce bands a little over 500bp. (Numbers on gel correspond to numbers on master plate.)
Everything as expected. New primer works well at 55°C.
Lane 1: Colony 1
Lane 2: Colony 2
Lane 3: Colony 3
Lane 4: Colony 4
Lane 5: Colony 5
Lane 6: 1 kb plus ladder
Lane 7: Colony 6
Lane 8: Colony 7
Lane 9: Colony 8
Lane 10: Colony 9
Lane 11: Colony 10
Lane 12: control
Ligations/Transformations 09/26
25min RE digests (re)done on appropriate minipreps using Fermentas enzymes and extracted using Clonewll gels.
We used the Takara ligation kit to do the following ligations (1:6 vector:insert):
- P38SP + P130XP
- P39SP + P130XP
- P38SP + P51XP
- P116SP + P130XP
- P108SP + P130XP
- P117SP + P130XP
- P51EX + P38ES (1/20)
- P51EX + P38ES (1/100)
- P51EX ligation control
We followed the Takara 25°C protocol and transformed all of the ligations into DH5α. We also let some of the ligations run overnight (16 °C for 8 hours -->12 °C for 4 hours --> 4 °C for eternity).