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Team:Bielefeld-Germany/Labjournal/week14
From 2012.igem.org
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| - | * '''Team Site Directed Mutagenesis:''' | + | * '''Team Site Directed Mutagenesis:''' Picked four colonies per dish ''T.vers.''_243 & ''T.vers.''_1161 and plated them for plasmid-isolation. |
| + | **Plasmid-isolation of ''B.pumi'' III(III)+IV(IV) with SDM-Mix 2317 ''X.camp' | ||
| + | **Test-restriction 700ng in 10 µL with PstI | ||
| + | Results of cutting if mutation failed: | ||
| + | The ''B.pumilus'' site directed mutation at 2317 is not a illegal restriktion site, so it can not be checked via Restriktion, but only through sequencing. | ||
| + | **Picked a few more Colonies of X.camp for plasmid-isolation | ||
===Wednesday August 1st=== | ===Wednesday August 1st=== | ||
Revision as of 21:43, 19 August 2012
Contents |
Labjournal
Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18
Week 14 (07/30 - 08/05/12)
Monday July 30th
- Team Site Directed Mutagenesis: Plasmid-isolations of plated colonies
- Team Cellulose Binding Domain: Redesigned the primer-sequences another time giving the CBDs a few AS more in than the Protein-BLAST said. CBDcex: 4 AS N-terminal, 2 C-terminal. CBDclos: 2 N-terminal (starting with a natural ATG) and 2 AS C-terminal.
- Prepared BBa_392014 for Sequencing
Tuesday July 31st
- Team Site Directed Mutagenesis: Picked four colonies per dish T.vers._243 & T.vers._1161 and plated them for plasmid-isolation.
- Plasmid-isolation of B.pumi III(III)+IV(IV) with SDM-Mix 2317 X.camp'
- Test-restriction 700ng in 10 µL with PstI
Results of cutting if mutation failed: The B.pumilus site directed mutation at 2317 is not a illegal restriktion site, so it can not be checked via Restriktion, but only through sequencing.
- Picked a few more Colonies of X.camp for plasmid-isolation
Wednesday August 1st
- Team Site Directed Mutagenesis: Plasmidisolation of T.vers_243 (I-IV) & T.v._1161 (I-IV) X.camp_2K&3K (V-VI)
- Test-restriktion with PstI / SpeI & Anaylsis via Gelelectrophoresis show bands as they should for T.vers_243 II+III; and X.camp._2K_2 V
- Made X.camp._2K_2 V and the final B.pumilusplasmids ready for sequencing
Thursday August 2nd
- Team Wiki: This morning we met for taking a new picture of our group and individual pictures of everyone. Check out our beautiful team members here.
- Team Site Directed Mutagenesis: SDM PCR of T.vers243 II+III with 1161 Primer mix and X.camp._2K_2 V with 3633
Friday August 3rd
- Team Site Directed Mutagenesis: Transformed T.vers_243_III_1161 and X.camp._2K_2 V_3633 in XL1 Blue and plated it on Ampicillin- and CM-selection-agar.
- Team Cellulose Binding Domain: Transformed the plasmids p714_cex and 570_clos we got from the fermantationgroup in KRX an plated them on Kanamycin-selectionagar.
Saturday August 4th
- Team Site Directed Mutagenesis: Plated four isolated colonies from T.vers_243_III_1161 and X.camp._2K_2 V_3633 per petri dish on seperated selection-agar-dishes
- Team Cellulose Binding Domain: Since there were only a few colonies on the p570 selection-agar-dish, I picked one, resuspended it in 100 µL destilled water and plated it on an new Kanamycin-selection-agar-dish
- plasmid-isolation of p714
Sunday August 5th
- Team Site Directed Mutagenesis: Plasmidisolations of X.camp.2K_2_3633 (I-IV) and T.vers_243_III_1161 (I-IV)
- Test-Restriktion of X.camp.2K_2_3633 (I-IV) and T.vers_243_III_1161 (I-IV) X.camp III + IV good; T.vers, no positiv, since the 1150 band should at least rise to 1550
- Picked two additional colonies of e.coli_2307 and plated them on CM
- Prepared X.camp.2K_2_3633 III + IV for sequencing
- Team Cellulose Binding Domain: Transformation of BBa_I13522 + pSB1A2 in KRX and plating it on AMP-Selection-Agar
- Plasmidisolations of p570
- PCR of CBDcex (417 bp) and CBDclos (369 bp)
- Test-Gelelectrophoresis: Got the bands that should be there!
- PCR Clean up (no Gelelectrophoresis)
- Restriktion of PCR-products and pSB1C3_RFP with XbaI and PstI.
