Gel substances
We receive all expected fragments.
Assembly of biobricks is successful.
The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
The samples were incubated for 1 h at 37° C.
Worked as expected.
3 out of 6 samples positive.
Sequencing pSB1A3 iBB486476315 K1 with primer 33 (iBB8 fw) AGBOOOK 275 and primer 34 (iBB8 rv) AGBOOOK 276.
Upper gel
Lower gel
The upper gel showed the expected bands, whereas these bands were missing in the lower gel.
Due to unreliabilities of the chain stop method by Sanger at the end of a DNA sequence, the detected gap can be considered as uncertain. This thesis is supported by 100% identity in the complementary strand.
The sequenced biobrick was believed to the accurate.
Several clones of pSB1A3-iBB486476315/-iBB496315/-iBB395416 were picked and inoculated in 100 ml LB medium containing ampicillin.
Cultures were incubated ON at 37 °C.
Midiprep of pSB1A3 iBB486476315, pSB1A3 iBB496315 and pSB1A3 iBB395416 (“QIAprep Spin Midiprep Kit” (Qiagen, Düsseldorf)).
Sample:
Digestion of 5 µl iBB4 with SpeI and PstI.
Digestion of 2 µl iBB10+9 and 4 µl iBB13+9 with XbaI and PstI.
The samples were incubated for 2 h at RT.
Transformation of the ligations into E. coli DH5α (30 min ice, 60 sec 42°C, 60 min 37°C + 900 µl LB), plated on LBCM, oN 37°C.
4 colonies of pSB1C3-iBB4+iBB10+iBB9 and 4 colonies of pSB1C3-iBB4+iBB13+iBB9 were inoculated in 5 ml LB medium containing chloramphenicol.
All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
The samples were incubated for 2h at 16 °C.
The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates. The plates were incubated over night at 37° C.
8 new sequencing samples were sent out.
4 colonies of pSB1A3-iBB4+iBB11+iBB96315 and 4 colonies of pSB1A3-iBB4+iBB12+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.
For making new aliquots of E. coli DH5α cells 50 ml LB medium were inoculated with 500 µl of an E. coli DH5α culture.
Two expected fragments are present. The relevant fragments are cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
The samples were incubated for 1h at 16 °C.
Resulting in the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.
An expected double band is shown in Sample 1 (Vector + Insert). The other sample do not exhibit the expected band and are therefore rejected.
About 80 new aliquots were made.
Expectations
4 colonies of pSB1A3-iBB4+iBB10+iBB96315 and the only single colony of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing chloramphenicol.
The chemo competent E. coli DH5α cells were transformed with the plasmid pSB1A3-iBB49+iBB6315 and plated on LB-Amp-plates. The plates were incubated over night at 37° C.
The culture of pSB1A3-iBB4+iBB13+iBB96315 had a pinkish color and therefore was discarded. The plasmid pSB1A3-iBB4+iBB10+iBB96315 was isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.
8 samples were sent out for sequencing.
Both colonies of pSB1A3-iBB4+iBB13+iBB96315 were inoculated in 5 ml LB medium containing ampicillin.
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