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    Week 33 (DNA)
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    Week 33 (DNA)

    Monday, 8/16/2021Plasmid prep for sequencingTable1Mix2Seq kitTable2Tuesday, 8/17/2021Plasmid prep of PET11Table3GelRestriction enzyme digestion and dephosphorylationGel electrophoresisTable4 Wednesday, 8/18/2021Restriction enzyme digestion and dephosphorylationGel electrophoresis Gel purification/plasmid cleanupTable5Concentration measurement of double digested plasmidTable6PCRPCR cleanupThursday, 8/19/2021Concentration measurement of inserts with PCR introduced RE sitesDigestion with RE of insertsGel electrophoresis of digestion with RE of insertsTable7Clean up of gel, cutting and purifyingTable8Ligation for RE cloningRecipesTable9Table10ProcedureTransformation and ON cultureFriday, 8/20/2021Checking the plates from the ON culture
    Monday, 8/16/2021
    Plasmid prep for sequencing
    Six plasmid preps were prepared using overnight incubation from the previous day using the E.Z.N.A plasmid prep kit. The following samples were used.
    1.
    CsgC 1:3
    2.
    CsgC 1:5
    3.
    DB3 1:3
    4.
    DSB 1:5
    5.
    PC23
    6.
    NC RE
    The protocol was followed with the following comments/adjustments:
    ●
    From the O/N cultures, three tubes each containing 10 ml were pooled into a 50 ml falcon tube resulting in a final volume of 30 ml.
    ●
    1.5 ml of solution I was added (3 times the volume of low copy plasmid)
    ○
    This was then divided into 6 eppendorf tubes, each containing approx. 250 µl.
    ■
    250µl and 350µl of Solution II and solution III respectively was to each of the eppendorf tubes
    ●
    After the 10 min centrifugation, the supernatant from the 6 eppendorf tubes were pooled.
    ●
    Samples were washed twice
    ●
    When eluting the plasmids, 30µl elution buffer was used. After that, the elution was repeated with an additional 30µl resulting in a final volume of 60µl eluted plasmid.
    NOTE! For the PC23 plasmid preparation we added solution III before solution II. A smaller cell debris pellet was observed compared to the other samples. Despite that, we decided to continue.
    Concentration measurement of plasmids. Elution buffer was used as blank.
    Mix2Seq kit
    Followed the protocol provided by eurofins except that the primer concentration was 10 times higher (100µM instead of the stated 10µM). Samples were sent to sequencing despite this mistake.
    Samples were
    ●
    PC 23: from plasmid prep (PP) 16/8. Conc = 73.1 ng/µl → direct
    ●
    Plasmid 0720: from PP 20/7. Conc = 132 ng/µl → 2x
    ●
    DB3 3:1: from PP 16/8. Conc =312 ng/µl → 5x
    ●
    DB3 5:1: from PP 16/8. Conc = 251 ng/µl → 3x
    ●
    CsgC 3:1: from PP 16/8. Conc = 207 ng/µl → 3x
    ●
    CsgC 5:1: from PP 16/8. Conc = 236 ng/µl → 3x
    Primers used were:
    ●
    SequencingEagI_Rev
    ●
    SequencingBamHI_Forw
    Identification number for each sample
    Tuesday, 8/17/2021
    Plasmid prep of PET11
    Four times 30 mL plasmid preps were prepared using the overnight incubation of E. coli PET11 from the previous day. pET11 is a high copy plasmid. However, the plasmids were prepared as if they were low copy plasmids, meaning that twice the volume of solution I, II and III was added.
    Concentration of plasmid prep
    Gel
    An agarose gel (0.6%) was prepared to verify the plasmid preparation and the enzyme digestion. For this purpose 1.2 g of agarose was dissolved in 200 mL of 1X TAE buffer by placing it into the microwave at max watt for 4 minutes. Subsequently the mixture was cooled down to 50 °c and 10 µl of Gel red was added.
    Restriction enzyme digestion and dephosphorylation
    The pET11 plasmid was digested with BamHI and NdeI, both separately and together. One sample wasn’t digested at all. The NdeI worked for 50 minutes. AP dephosphorylation enzyme was present during the full reaction. It should have been added after the digestion was complete. Followed the restriction enzyme digest protocol found on ThermoFisher.
    Gel electrophoresis
    Loading scheme
    pET11 - from plasmid prep 2021-08-17 conc. = 303 ng/µl.
    “-” - dephosphorylated pET11 plasmid. Non digested
    BamHI - dephosphorylated pET11 plasmid digested with BamHI
    NdeI - dephosphorylated pET11 plasmid digested with NdeI
    Both - dephosphorylated pET11 plasmid digested with both BamHI and NdeI
    At first wells 1-7 were loaded and ran for an hour at 90V.
    Wells 9-11 were added later and ran for in total 50 min at 90V meaning that wells 1-7 ran for a total of almost 2 hours.
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    ●
    pET11 had two clear bands. The lower represents the supercoiled form. The upper band is the open circular or linerized version of the vector.
    ●
    The dephosphorylated plasmid “-” should have the same bands as the regular pET11 vector which it does. However, the bands are fainter.
    ●
    The BamHI dephosphorylation did not seem to work well since we expect it to have one band only (linearized form). The plasmid could still appear in its supercoiled form.
    ●
    The NdeI digestion worked out better since no supercoiled form of the plasmid
    ●
    Same goes for the double digested plasmid. Or does this just represent the effect of NdeI?
    After trying out the BamHI (old and new tube) another time, only one band was visible suggesting that the digestion was successful. However, the bands were very faint and it’s difficult to distinguish the bands from the ladder.
    Wednesday, 8/18/2021
    Restriction enzyme digestion and dephosphorylation
    6*20µl reactions (restriction enzyme digestion and subsequent dephosphorylation) were performed with the plasmid extracted from the previous day (tube 1, 303 ng/µl). The following was added into each eppendorf tube:
    ●
    Sterile water (12 µl)
    ●
    10X buffer (2µl)
    ●
    Plasmid (3µl, 303 ng/µl)
    1µl NdeI was added to the reaction mix and placed on a heat block at 37ºC for 40 minutes. 1µl BamHI was added to the reaction mix after 40 minutes and left on a heat block at 37ºC for another 10 minutes.
    Restriction enzyme reactions were then terminated by placing on a heat block at 80ºC for 5 minutes and left to cool. 1µl fastAP was then added to the reaction mixture and left on a heat block at 37ºC for another 10 minutes for dephosphorylation.
    Gel electrophoresis
    An agarose gel (0.6%) was prepared to verify the plasmid preparation and the enzyme digestion. For this purpose 1.2 g of agarose was dissolved in 200 mL of 1X TAE buffer by placing it into the microwave at max watt for 4 minutes. Subsequently the mixture was cooled down to 50 °C and 10 µl of Gel red was added.
    Gel electrophoresis was performed with 1kb ladder and all the contents from the restriction enzyme digestion (each 20µl reaction was mixed with 4µl loading dye). Gel electrophoresis was run at +60 V for 5 minutes and +90 for 1 and half hours. Gel doc was used for visualization and subsequent gel purification.
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    Gel purification/plasmid cleanup
    6 eppendorf tubes were preweighed for the gel purification. The bands identifying the digested plasmid were cut out and placed into the eppendorf tubes and the tubes were weighed again for determination of gel volume. The weight of the tubes before and after, as well as amount of buffer to be added for gel purification is shown below:
    Gel purification of the double digested plasmid was performed according to the protocol given in the nucleospin kit with the following comments/changes
    ●
    Samples were washed twice with NT3 buffer.
    ●
    NE buffer (elution buffer) was preheated to 70°C. 15 µl was added to the column, incubation for 5 min at RT and then centrifuged according to protocol. This was repeated one more time resulting in a final volume of 30 µl. The tubes were put in the freezer
    Concentration measurement of double digested plasmid
    PCR
    Mastermix was prepared according to the following recipe:
    ●
    228.6 µl H2O
    ●
    60 µl phusion HF buffer
    ●
    100M dNTP (0.6 µl each)
    ●
    3 µl phusion polymerase
    The mastermix was aliquoted into 5 PCR tubes, 49 µl in each. Subsequently the specific template with suitable primers were added. 0.25 µl of each primer, and 0.5µl of template.
    Following primers were used
    ●
    DB3DB3: DB3_For1, DB3_Rev1
    ●
    tANK6: tANK_NdeI, tANK_BamHIrev
    ●
    CsgC: CsgC_IntNdeI_For, CsgC_BamHI_Rev1
    ●
    CsgF: CsgF_For1 (NdeI), CsgF_Rev1 (BamHI)
    ●
    degP: degP_IntNdeI_For, degP_IntBamHI_Rev
    Following templates were used (Slpmod):
    ●
    DB3DB3
    ●
    tANK6
    ●
    CsgC
    ●
    CsgF
    ●
    degP
    **NOTE: primer DB3_For1 was opened before spin down. Some translucent film/powder might have been lost
    PCR cleanup
    Was done according to protocol.
    Thursday, 8/19/2021
    Concentration measurement of inserts with PCR introduced RE sites
    The concentrations in ng/µl of the inserts were the following:
    ●
    DB3DB3: 145
    ●
    tANK6:  109
    ●
    CsgC:  149
    ●
    CsgF: 309
    ●
    degP: 90,5
    Digestion with RE of inserts
    Was performed with NdeI (50 min) and BamHI (10 min) according to protocol.
    Gel electrophoresis of digestion with RE of inserts
    Was done according to protocol. 1% agarose gel. Added 24 µl of each insert + dye in the respective well.
    Loading scheme:
    Clean up of gel, cutting and purifying
    The clean up was done according to protocol. The concentrations of the inserts were; DB3DB3 30.3 ng/µL, tANK6 20.4 ng/µL, CsgC 19.4 ng/µL, CsgF 18.4 ng/µL and DegP had a concentration of 23.4 ng/µL.
    Ligation for RE cloning
    Insert to vector molar ratios 3:1. 50 ng vector was used. Recipes and procedure below were followed
    Note! 10 aliquotes with 50 µL 10x T4 ligase buffer were made and stored in freezer.
    Recipes
    For all samples
    For each sample
    Procedure
    1.
    Thaw on ice, briefly vortex and briefly spin down all solutions
    2.
    Prepare reaction mixtures following recipes above
    a.
    Add ligase last
    b.
    Mix by pipetting up and down
    c.
    Briefly vortex and spin down to mix fully
    3.
    Incubate 2 hours at room temperature
    Transformation and ON culture
    Done according to protocol found in the physical lab notebook. 400 µl LB media was added for cell recovery, and in order to concentrate the cells, 529µl was removed after centrifugation and the cell pellet was resuspended in remaining media. 50µl of the resuspension was then spread on LB agar plates containing 100 µg/ml ampicillin.
    Friday, 8/20/2021
    Checking the plates from the ON culture
    The plates all had a carpet of bacterial growth, including the negative control, suggesting that the ampicillin selection did not work, most likely due to the ampicillin not working properly.

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