Team:CityU HK/notebook/lablog
From 2014.igem.org
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 by miniprep
- Restriction digestion of fadL, fadD inserts by XbaI and PstI & BBa_R0040, BBa_B0030 (rbs) by SpeI and PstI
-> No band is observed for BBa_B0030 after digestion
-> Gel photo showing extracted BBa_B0030 is contaminated with RNA
-> Lead to overestimation of BBa_B0030 concentration for digestion- Purification of digested fadL, fadD inserts, BBa_R0040 products
- Ligation of digested fadD insert into BBa_B0030 vector
- DH5α competent cell is transformed with pSB1C3_ R0040-fadL by heat shock
- Repeat extraction of BBa_B0030 by miniprep, product is treated with RNase A
- Restriction digestion of Bba_B0030 by SpeI and PstI
- Purification of digested Bba_B0030
- Ligation of fadL insert into BBa_R0040 vector
- LB plate preparation with Chloramphenicol (34ug/ml)
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Early AugustOpen or Close
Week 1 (3/8~9/8)
- DH5α competent cell is transformed with pSB1C3_ B0030-fadD by heat shock
- Screening for pSB1C3_ R0040-fadL clones by colony PCR
-> non-specific bands observed- Screening for pSB1C3_ B0030-fadD clones by colony PCR
-> non-specific bands observed- Repeat screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 2 clones and sent for sequencing- Repeat screening for pSB1C3_ B0030-fadD clones by colony PCR
-> Bands with expected size are observed in 6 clones and sent for sequencingWeek 2 (10/8~16/8)
- Sequencing results show negative result for the sent clones:
-> Missing one to two base pair in fadL gene in pSB1C3_ R0040-fadL clones
Missing ribosomal binding site in pSB1C3_ B0030-fadD clones- Repeat screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 4 clones and sent for sequencing- PCR amplification of fadD, fadL inserts with 1°C higher annealing temperature
- Purification of fadD, fadL PCR products
- Restriction digestion of fadL, fadD inserts by XbaI and PstI
- Extraction of BBa_B0034 (biobricks with ribosomal binding site) by miniprep
- Extraction of BBa_R0040 by miniprep
- Restriction digestion of BBa_R0040 by SpeI and PstI
- Purification of digested BBa_R0040 product
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Late AugustOpen or Close
Week 3 (17/8~23/8)
- Sequencing results show negative result for the sent clones last week:
-> Missing one to two base pair in fadL gene in pSB1C3_ R0040-fadL clones- Ligation of fadL insert into BBa_R0040 vector
- DH5α competent cell is transformed with pSB1C3_ R0040-fadL by heat shock
- Restriction digestion of BBa_B0034 by SpeI and PstI
- Purification of digested BBa_B0034 product
- Ligation of fadD insert into BBa_B0034 vector
- LB plate preparation with ampicilin (100ug/ml) for selection of pSB1A2 clones
- DH5α competent cell is transformed with pSB1A2_ B0034-fadD by heat shock
- Screening for pSB1C3_ R0040-fadL clones by colony PCR
-> Bands with expected size are observed in 1 clones and sent for sequencingWeek 4 (24/8~30/8)
- Screening for pSB1A2_ B0034-fadD clones by colony PCR
-> Bands with expected size are observed in 4 clones and sent for sequencing- PCR amplification of fadD, fadL inserts with ?°C higher annealing temperature
- LB plate preparation with ampicilin (100ug/ml)