Team:CityU HK/notebook/lablog
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FadD & FadL Module's Lablog
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July - Week 4 (20/7~26/7)Open or Close
- PCR amplification of fadD, fadL inserts
- Purification of fadD, fadL PCR products
- LB plate preparation with chloramphenicol (34ug/ml) for selection of pSB1C3 clones
- DH5α competent cells are transformed with BBa_B0030 and BBa_R0040 separately by heat shock
- 1st Extraction of BBa_B0030, BBa_R0040 by miniprep
-> low product yield of BBa_R0040- Extraction of BBa_R0040, by miniprep
-> low product yield- New batch of BBa_R0040 transformants are prepared in LB broth
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July - Week 5 (27/7~2/8)Open or Close
- Extraction of BBa_R0040 were performed with plasmid miniprep kit
- Restriction digestion were performed on:
fadL, fadD inserts with XbaI and PstI
BBa_R0040, BBa_B0030 with SpeI and PstI
Results: No band was observed for BBa_B0030 after digestion
-> Gel photo showed the extracted BBa_B0030 DNA was contaminated with RNA leading to overestimation of BBa_B0030 concentration for restriction digestion- Purification was performed on the digested fadL and fadD inserts and BBa_R0040 products
- Ligation of digested fadD insert into BBa_B0030 vector was performed
- DH5α competent cell was transformed with pSB1C3_ R0040-fadL by heat shock protocol
- BBa_B0030 was performed again with plasmid DNA miniprep kit, the product was treated with additional RNase A
- Restriction digestion of BBa_B0030 was performed with SpeI and PstI
- Digested BBa_B0030 was purified with PCR purification kit
- fadL insert was ligated into BBa_R0040 vector
- LB plate with chloramphenicol (34 µg/mL) was prepared
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Early AugustOpen or Close
Week 1 (3/8~9/8)
- DH5α competent cells were transformed with pSB1C3_ B0030-fadD by heat shock protocol
- pSB1C3_ R0040-fadL clones was screened with colony PCR method
-> non-specific bands were observed- pSB1C3_ B0030-fadD clones were screened by colony PCR method
-> non-specific bands were observed- Repeated colony PCR analysis on the pSB1C3_ R0040-fadL clones
-> Bands with expected size were observed in two clones and sent for DNA sequencing- pSB1C3_ B0030-fadD clones were screening again with colony PCR method
-> DNA Bands with expected size were observed in 6 clones and sent for DNA sequencingWeek 2 (10/8~16/8)
- Sequencing results show that the transformants contained errors in DNA sequences
-> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL clones
-> Missing the ribosomal binding site in the pSB1C3_ B0030-fadD clones- Repeat screening for pSB1C3_ R0040-fadL clones were performed with the colony PCR method
-> Bands with expected size were observed in 4 clones and sent for DNA sequencing- PCR amplification of fadD, fadL genes with higher annealing temperature
- Purification of fadD, fadL PCR products
- Double restriction digestion on fadL, fadD PCR products with XbaI and PstI
- Extraction of BBa_B0034 (biobricks with the ribosomal binding site) plasmid by plasmid DNA miniprep kit
- Extraction of BBa_R0040 plasmid with plasmid DNA miniprep kit
- Double restriction digestion on BBa_R0040 plasmid with SpeI and PstI
- Purification of the digested BBa_R0040 plasmid
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Late AugustOpen or Close
Week 3 (17/8~23/8)
- Sequencing results showed errors in DNA sequences in the clones prepared last week
-> Missing one to two base pairs in the fadL gene in the pSB1C3_ R0040-fadL plasmid- Ligation was performed on the fadL insert and the BBa_R0040 vector
- DH5α competent cell was transformed with the pSB1C3_ R0040-fadL plasmid by the heat shock protocol
- Double restriction digestion on BBa_B0034 by SpeI and PstI
- Purification of the digested BBa_B0034 plasmid
- Ligation were performed on the fadD insert and the BBa_B0034 vector
- LB plate with ampicillin (100 µg/mL) were prepared for the selection of pSB1A2 clones
- DH5α competent cells were transformed with pSB1A2_ B0034-fadD by the heat shock protocol
- Screening for pSB1C3_ R0040-fadL clones were done by the colony PCR method
-> DNA bands with expected size were observed in only one clone and sent for DNA sequencingWeek 4 (24/8~30/8)
- Screening on the pSB1A2_ B0034-fadD clones were done by the colony PCR method
-> DNA bands with expected size were observed in 4 clones and sent for DNA sequencing- PCR amplification of the fadD, fadL inserts were performed at higher annealing temperature
- LB plates with ampicillin (100 µg/mL) were prepared
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September - Week 1 (31/8~6/9)Open or Close
- Sequencing results:
The identity of the DNA insert in pSB1C3_ R0040-fadL clone was confirmed by DNA sequencing.
Changes in DNA sequence were found in the fadD gene in pSB1A2_ B0034-fadD clones
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