Team:Vilnius-Lithuania/Labjournal

About

pLac+cI.
P10 and P11 plasmids are used. pLac (P11) and repressor cI (P10) ligation.

Date Materials Experiment
06 30 P10
P11		 P10 and P11 transformation into DH5α.
07 01 Bacteria inoculation (4 minipreps).
07 09 P10
P11		 Plasmids’ restriction (3 ug each).
P10 with EcoRI and XBal (+FastAp).
P11 with EcoRI and SpeI.
Extraction from gel
L10 and L11 are produced.
Conc.: L11 – 2,4 ng/uL.

Conclusions and Results:

07 14 – all of the colonies are successful, except third colony.

About

Cas3 with mutated restriction sites
L7+L15. Concentration - 261 ng/μL

Date Materials Experiment
06 30 P13
P7		 P13 and P7 transformation into DH5α.
07 01 Transformants are inoculated (4 minipreps each).
07 02 Plasmid extraction. Concentrations:
P7 – 174,1 ng/μL and 166 ng/μL.
P13 - 216 ng/μL and 131, 5 ng/μL.
07 13 P13 Cas3 mutagenesis (with ST1/2, ST3/4, ST5/6 primers).
07 16 Mutated P13 Cas3 plasmids extraction.
07 21-23 Mutated P13 Restriction with EcoRI, XbaI and PstI.
07 27 Mutated P13 Cas3 mutagenesis.
07 29 P7 Restriction with EcoRI and SpeI. L7 is produced.
07 31 P13
08 02 Mutated P13 Mutated Cas3 restriction with EcoRI, XbaI and PstI.
08 05 Mutated P13 Cas3 mutagenesis.
08 07 Mutated P13 Mutated Cas3 restriction with PstI. Mutagenesis was successful.
L15 is produced.
08 11 L7
L15		 L7 and L15 ligation with pSB1C3 (EcoRI+XBal+FastAp).
P15.1 is produced.
P15.1 Sequencing.

Conclusions and Results:

07 21 – Mutagenesis was unsuccessful.
08 02 – successfully mutagenized EcoRI and XbaI sites.
08 07 – successful mutagenesis confirmed (XbaI and EcoRI sites are mutated).
08 07 – Successful mutagenesis (PstI, XbaI and EcoRI sites are mutated).

About

P26 – L7+L26 pLux/cI+SRBS
P27 – L7+L27 pLux/cI+MRBS
P28 – L7+L28 pLux/cI+WRBS
P29 – L7+L29 pLux/cI

Date Materials Experiment
07 27 ST9
ST10
ST11
ST12
ST15
ST16
ST17
ST18
ST19
ST20		 pLux/cI contruction from oligos – PCR reaction with ST9 and ST10/11/12 primers, then with
ST15 and ST16/17/18.
Parallel PCR with ST9 and ST19 and then with ST15 and ST20.
PCR products purification.
07 28 PCR products from oligos restriction with EcoRI and SpeI.
L26, L27, L28 and L29 are produced.
07 28 L7
L26
L27
L28
L29		 Ligation of:
L7 and L26, L7 and L27, L7 and L28, L7 and L29
and their transformation into DH5α.
07 29 -
- 08 05		 L7+L26 colony PCR with ST15 and VR.
L7+L27 colony PCR with ST15 and VR.
L7+L28 colony PCR with ST15 and VR.
1st and 2nd colonies are inoculated.
P26, P27, 28 and P29 are produced
P26, P27, 28 and P29 restriction with NspI.
08 10 P26, P27, 28 and P29 sequencing with VF2.

Conclusions and Results:

07 31 – all of the colonies are successful.
08 10 – all biobricks are sequenced succesfully.

About

pLac+cI.
P10 and P11 plasmids are used. pLac (P11) and repressor cI (P10) ligation.

Date Materials Experiment
06 30 P10
P11		 P10 and P11 transformation into DH5α.
07 01 Bacteria inoculation (4 minipreps).
07 09 P10
P11		 Plasmids’ restriction (3 ug each).
P10 with EcoRI and XBal (+FastAp).
P11 with EcoRI and SpeI.
Extraction from gel
L10 and L11 are produced.
Conc.: L11 – 2,4 ng/uL.

Conclusions and Results:

07 14 – all of the colonies are successful, except third colony.

About

pLac+cI.
P10 and P11 plasmids are used. pLac (P11) and repressor cI (P10) ligation.

Date Materials Experiment
06 30 P10
P11		 P10 and P11 transformation into DH5α.
07 01 Bacteria inoculation (4 minipreps).
07 09 P10
P11		 Plasmids’ restriction (3 ug each).
P10 with EcoRI and XBal (+FastAp).
P11 with EcoRI and SpeI.
Extraction from gel
L10 and L11 are produced.
Conc.: L11 – 2,4 ng/uL.

Conclusions and Results:

07 14 – all of the colonies are successful, except third colony.