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    Week 29 (DNA)
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    Week 29 (DNA)

    Monday, 7/19/2021Material preparations (to use on the same day)Agarose gel (200 ml, diluted in TAE) for plasmid visualisationMaterial preparations (to use on the next day)Preparation of plasmid extractionSimultaneous enzyme digestion and dephosphorylationGel electrophoresis DNA extraction from agarose gelsTable1Tuesday, 7/20/2021Plasmid prepTable2Gel electrophoresisPreparation of new erythromycin stock solutionPreparation of LB solid medium and plates with 100 ug/ml erythromycinTransformation of E.coli with Gibson assembly constructs (Old homology)Wednesday, 7/21/2021Plate observationsAgarose gel (200 ml, diluted in TAE) for plasmid visualisationGel electrophoresisTable3 Transformation of E.coli with Gibson assembly constructs (Old homology)Plate observations 2Thursday, 7/22/2021Plate observationsAgarose gel (400 ml, diluted in 100*TAE) for plasmid visualisationColony PCRGel electrophoresis on PCR productsTable4 Table5 Master platesFriday, 7/23/2021Observations on master platesAgarose gelGel electrophoresis on PCR productsTable6 LB-agar plates
    Monday, 7/19/2021
    Material preparations (to use on the same day)
    Agarose gel (200 ml, diluted in TAE) for plasmid visualisation
    0.6% agarose 1.2g
    20000*  gelred 10ul
    Note: TAE stock solution buffer should be diluted 100 times when making a gel
    Material preparations (to use on the next day)
    Preparation of LB solid medium and plates with 100 ug/ml erythromycin
    Prepared according to ​LB agar plates recipe, two batches with 100 ug/ml erythromycin. The erythromycin was added when the solution had reached body temperature before pouring in plates. Some agar solidified in the bottom of the flask, yielding 46 usable 100 ug/ml plates.
    Preparation of plasmid extraction
    Plasmid extraction was started and cultures will grow overnight. 12 culture tubes were made according to the protocol from E.Z.N.A.
    Simultaneous enzyme digestion and dephosphorylation
    2 reactions are performed using FEM50 plasmid. Simultaneous enzyme digestion (BamHI and EagI) is performed for both of them and for one of them an additional dephosphorylation is performed (FastAP Thermosensitive Alhakine Phosphatase).
    ●
    Extracted plasmid is digested with BamHI and EagI using the following recipe:
    ○
    14 µl extracted plasmid (Using FEM50 with conc 0.144 µg/µL, prepared 07/06/2021)
    ■
    2 µg DNA
    ○
    2 µl sterile water/ FastAP Thermosensitive Alkaline Phosphatase
    ■
    Make total reaction volume 20 µL
    ○
    2 µl 10x FD buffer (included in restriction digest kit)
    ○
    1 µl BamHI restriction enzyme
    ○
    1 µL EagI restriction enzyme
    ●
    Reactions were incubated at 37ºC for 10 minutes, and terminated at 80ºC for 20 minutes using different heating blocks
    The tubes are labelled “FEM50 d.dig” (not dephosphorylated)  and “FEM50 d.dig AP” (dephosphorylated). Both stored in the freezer.
    Gel electrophoresis
    Gel electrophoresis was done on 0.6% agarose gel(200ml, 10ul Gelred). The following amount of samples were added into the wells:
    ●
    2 µl 1kbp gene ruler
    ●
    5 µl F50（133 ng/ul)+1ul loading dye (negative control)
    ●
    10 µl BamHI and EagI digested FEM50 + 2ul loading buffer
    ●
    10 µl BamHI and EagI digested FEM50 dephosphorylated + 2ul loading buffer
    ●
    6 µl BamHI digested FEM50 (mixed with loading dye)
    Gel electrophoresis was performed at 60 V for 5 minutes and 90 V for 60 minutes. Visualisation on geldoc showed that plasmid prep and restriction enzyme digest was successful for all samples.
    Loading...
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    DNA extraction from agarose gels
    Check MACHEREY-NAGEL PCR clean-up Gel extraction User manual as the standard protocol.
    Concentration measured with lid 10
    BamHI and EagI digested FEM50 - sample1
    10 µl BamHI and EagI digested FEM50 dephosphorylated - sample2
    Transformation of E.coli (test for competent cells XL1-Blue)
    ●
    Take out plates with proper antibiotics
    ○
    Note! Used plates 50 ug/ml erythromycin plates from 20210622 and 100 and 150 ug/ml erythromycin plates from 20210716
    ●
    Thaw the competent cells on ice for 30 min
    ●
    Add 1ul 72.1ng/ul plasmid pTRKH3-ermGFP and mix gently
    ○
    Marked FEM10 in freezer
    ●
    Incubate on ice for 40 min
    ●
    Heat shock the cells at 42℃ for 60s
    ●
    Place the cells on ice for 2min
    ●
    Add 400 ul LB medium and incubate at 37℃ for 45min
    ●
    Take 100ul out from the tubes and spread out the cells on LB plates
    ○
    Totally 10 plates, a positive and a negative control (no plasmid) for each concentration of erythromycin, one undiluted sample and one concentrated x3. 50ug/ml erythromycin concentrated negative control and 150ug/ml erythromycin concentrated positive control were not done due to a lack of sample.
    ○
    Concentration was done by centrifuging samples for 2 min at 4000rpm then removing 200 ul supernatant, then resuspending in the remaining 100ul supernatant
    ●
    Incubate plates at 37℃ overnight
    Second Transformation of E.coli (test for competent cells XL1-Blue)
    ●
    Take out plates with proper antibiotics
    ○
    Note! Used plates 100 ug/ml erythromycin plates prepared today
    ●
    Thaw the competent cells on ice for 30 min
    ●
    Add 1ul 72.1ng/ul plasmid pTRKH3-ermGFP and mix gently
    ○
    Marked FEM10 in freezer
    ●
    Incubate on ice for 40 min
    ●
    dHeat shock the cells at 42℃ for 60s
    ●
    Place the cells on ice for 2min
    ●
    Add 400 ul LB medium and incubate at 37℃ for 55min
    ●
    Take 100ul out from the tubes and spread out the cells on LB plates
    ○
    Totally 4 plates, a positive and a negative control (no plasmid) one undiluted sample and one concentrated x5
    ■
    Concentration was done by centrifuging samples for 2 min at 4000rpm then removing 400 ul supernatant, then resuspending in the remaining 100ul supernatant
    ●
    Incubate plates at 37℃ overnight
    Overnight (ON) inoculation for Plasmid Prep
    ●
    12x10 ml tubes were prepared for inoculation of E. coli pTRKH3-ermGFP containing the addgene vector. All tubes contained LB media with 10 µg/ml erythromycin. Details of inoculation are as follows:
    ○
    12x10 ml E. coli pTRKH3-ermGFP streaked out on 50 µg/ml erythromycin plate (from original viall)
    ○
    Tubes were incubated at 37ºC on shake
    Tuesday, 7/20/2021
    Plasmid prep
    ●
    4 plasmid preps were performed on the ON inoculations prepared in the previous day (see material preparations on previous page for details)
    ●
    Results of dsDNA concentration of extracted plasmids are as follows (4 tubes are combined into 1)
    ●
    There is one tube add 350ul solution 1, 350ul solution 2 and 490ul solution 3.
    Gel electrophoresis
    ●
    Gel electrophoresis was done on 0.6% agarose gel. The following amount of samples were added into the wells:
    ○
    3 µl 1kbp gene ruler
    ○
    6 µl plasmid extracted today
    ○
    3.5ul plasmid(144.4ng/ul)
    ●
    Gel electrophoresis was performed at 60 V for 5 minutes and 90 V for 60 minutes. Visualisation on geldoc showed that plasmid prep and restriction enzyme digest was successful for all samples.
    ●
    The well of the gel had some problems and sample 2 disappeared and sample 1 and 3 didn’t run in the gel.
    Preparation of new erythromycin stock solution
    250 mg erythromycin is dissolved in 5 ml EtOH to make stock solution with concentration 50 mg/ml.
    Preparation of LB solid medium and plates with 100 ug/ml erythromycin
    Prepared according to ​LB agar plates recipe x2, 48 plates in total.  New erythromycin stock solution prepared above was used. The only thing that did not follow the protocol was that some solidified bits in the bottles were formed before we were ready to pour them so we reheated them in the autoclave and then everything followed the protocol again.
    Transformation of E.coli with Gibson assembly constructs (Old homology)
    ●
    Thaw the competent cells on ice for 30 min
    ●
    Add 10ul plasmid and mix gently.
    ○
    Gibson assembly constructs:
    ■
    A10, A20, A30, A40, A50, A60, A80, A90, A100, A110
    ■
    Note! A70 could not be found
    ■
    Note! there were two marked A20. The A20 used was marke with “x”
    ○
    Positive control: undigested plasmid marked FEM10 in freezer
    ○
    Negative control: Negative control in the gibson assembly
    ●
    Incubate on ice for 40 min (Note time!)
    ●
    Heat shock the cells at 42℃ for 60s
    ●
    Place the cells on ice for 3min
    ●
    Add 400 ul LB medium and incubate at 37℃ for 55min (Note time!)
    ●
    Take out plates with proper antibiotics
    ○
    Note! Used plates 100 ug/ml erythromycin plates from yesterday, 20210719 and 100 ug/ml erythromycin plates from today 20210720
    ●
    Concentrate by centrifuging samples for 3 min at 4000rpm then removing 350 ul supernatant, then resuspending in the remaining 250ul supernatant
    ●
    Take 100ul out from the tubes and spread out the cells on LB plates
    ○
    In total 24 plates, 10 constructs, one negative control, one positive control. One old and one new plate for each construct and control
    ○
    Note! A100 100ug/ml EM prepped 19/7 had approximately half the volume (50ul) plated instead of 100ul
    ●
    Incubate plates at 37℃ overnight
    Note! Tubes of transformants were left in the fridge with approx 50 ul left if we want to plam them again
    Wednesday, 7/21/2021
    Plate observations
    Plates were incubated the wrong way (lid up). Very few colonies. Both positive controls were good with several colonies. Plates were put back into the incubator for further growth (right way).
    Agarose gel (200 ml, diluted in TAE) for plasmid visualisation
    0.6% agarose 1.2g
    20000*  gelred 10ul
    Gel electrophoresis
    ●
    Gel electrophoresis was done on 0.6% agarose gel. The following amount of samples were added into the wells:
    ○
    3 µl 1kbp gene ruler
    ○
    6ul plasmid(144.4ng/ul) （5+1 loading buffer)
    ○
    6 µl plasmid extracted yesterday（5+1 loading buffer)
    ●
    Gel electrophoresis was performed at 60 V for 5 minutes and 90 V for 60 minutes. Visualisation on geldoc showed that plasmid prep and restriction enzyme digest was successful for all samples.
    ●
    The plasmid extracted yesterday has some genome DNA pollution, but the band is the same with the previous one.
    Loading scheme:
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    Transformation of E.coli with Gibson assembly constructs (Old homology)
    ●
    Thaw the competent cells on ice for 30 min
    ●
    Add the following and mix gently.
    ○
    10 µL of each of the Gibson assembly constructs:
    ■
    A70, A120, A130, A140, A150, A160, A170, A180
    ○
    1 µL Positive control: undigested plasmid marked FEM10 in freezer
    ○
    5 µLNegative control: Negative control in the gibson assembly
    ○
    Note! The tube that was thought to be marked A20 but that was not used 20210720 turned out to be A70.
    ●
    Incubate on ice for 40 min (Note time!)
    ●
    Heat shock the cells at 42℃ for 60s
    ●
    Place the cells on ice for 3min
    ●
    Add 600 µl LB medium and incubate at 37℃ for 60 min.
    ○
    Note! Increased LB volume (originally 400µL)
    ○
    Note! For 4 of the tubes liquid TG1 culture was mistakingly added instead of LB medium. Uncertain which ones.
    ●
    Take out plates with proper antibiotics
    ○
    Note! Used plates 100 ug/ml erythromycin plates from yesterday, 20210720
    ●
    Concentrate by centrifuging samples for first 1 min 30 sec at 4100 rpm, then 1 min 5000 rpm.
    ●
    For the GA samples, 740 µL supernatant was removed, cells resuspended in the remaining 60 µL, all of which was used for inoculating the plates.
    ●
    For the controls, 680 µL supernatant was removed so that two plates could be inocculated with 60µL each.
    ●
    In total 12 plates, 10 constructs, one negative control, one positive control. O
    ●
    Incubate plates at 37℃ overnight
    Note! Tubes of transformants were left in the fridge with approx 50 ul left if we want to plam them again
    Plate observations 2
    Plates in incubator were checked again at 13:30. The following were put in the fridge to prevent overgrowth:
    ●
    Plates prepared 20210719: A10, A30
    ●
    Plates prepared 20210720: A10, A20, A30, A40, A80, A100, A110
    The remaining plates were put back into the incubator at 37°C.
    Thursday, 7/22/2021
    Plate observations
    Transformants A10-A60, A80-A110 on plates prepared 20/7, transformed 20/7, had enough colonies.
    Transformants A120-A130 on plates prepared 20/7, transformed 21/7, had enough colonies.
    Transformants A140-A180 on plates prepared 20/7, transformed 21/7, were suspected to be contaminated with TG1 cells.
    Transformant A70 on plates prepared 20/7, transformed 21/7,  had very few colonies.
    Transformants Negative Ligation mix on plates prepared 20/7, transformed 20/7 and 21/7 might be contaminated.
    Transformants F10 plasmid (positive control) on plates prepared 20/7, transformed 21/7 might be contaminated.
    Agarose gel (400 ml, diluted in 100*TAE) for plasmid visualisation
    Two agarose gels were prepared.
    1% agarose 4g
    20000*  gelred 20ul
    Colony PCR
    Protocol ​Colony PCR was followed to make 73 20µL reaction in total. 72 sections were streaked on ​Masterplates and 1 negative PCR control (no template DNA). MasterMix enough for 80 20µL reactions, was prepared to have margins, in total 1600 µL. Primers For2 and Rev2 were used, so annealing temperature in PCR was Tm - 5 = 56°C.
    Gel electrophoresis on PCR products
    Buffer was exchanged in the GE chamber in the course lab and in the GE chamber at division. Gels were run for 45 min at 90V instead of 60 min. Two gels were run. Visualisation in GelDoc and loading schemes are shown below. Note! Not enough wells for all PCR products, so some were put in the freezer to be run at a later date.
    Loading scheme for Gel 1:
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    Loading scheme for Gel 2:
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    Master plates
    3 of the master plates are deemed to have grown enough and placed in the fridge: 49-54, 55-60 and 61-66.
    The remaining plates are kept in the incubator at 37°C overnight.
    Friday, 7/23/2021
    Observations on master plates
    No growth on sections: 17, 23, 30, 58, 60, 72.
    Little growth on sections: 49, 50, 53, 54, 59.
    Agarose gel
    1% agarose gel, 200 mL.
    Gel electrophoresis on PCR products
    3 µL 1 kbp
    Loading scheme:
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    LB-agar plates
    42 LB-agar plates with 100 µg/mL erythromycin were made.

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