Team:CityU HK/notebook/lablog

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<div><img src="http://2014.igem.org/wiki/images/e/ee/CityU_HK_lablog.png" width="100%"></div>
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<h1 id="title">FadD & FadL Module's Lablog</h1>
<h1 id="title">FadD & FadL Module's Lablog</h1>

Latest revision as of 16:54, 17 October 2014

Bootstrap 101 Template

FadD & FadL Module's Lablog

  • 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

  • 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

  • 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 sequencing

    Week 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

  • 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 sequencing

    Week 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

  • 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