TITLE: Transformation of E.coli with pGem-T Easy
AUTHORS: Gustaf Degen

[DESCRIPTION]
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[STEPS]
?. [A-tailing procedure (Day 1)]
Because the Phusion polymerase will remove the A-overhangs, adding these with 
Taq DNA Polymerase is necessary, as this is required for cloningMix the following reagents:  AB1PCR Product1-7µl210x Taq Buffer1µl3ATP to 0.2mM1µl4Taq DNA Pol 5U1µl5dH2Oto 10µlIncubate for 30 min at 70°CIdeally and if DNA conc. is high engouh
(> 50ng/µl), do another quick cleanup with the Promega Kit.
AB1PCR Product1-7µl210x Taq Buffer1µl3ATP to 0.2mM1µl4Taq DNA Pol 5U1µl5dH2Oto 10µl
?. [Ligation  (Day 1)]
Calculate the ratio of Vector:Insert (use molar ratios of 1:1 or 3:1):Mix the following reagents:  AB1Vector (50ng/µl) 3kb50ng2Insertxng32x Rapid Lig. Buff.1µl4T4 Ligase1µl5dH2Oto 10µlIncubate at 4°C over night for maximum ligation efficiency or 3 hours at room temperature.
AB1Vector (50ng/µl) 3kb50ng2Insertxng32x Rapid Lig. Buff.1µl4T4 Ligase1µl5dH2Oto 10µl
?. [Transformation (Day 2)]
Thaw DH5⍺ on ice (100 µl in tube)Add 3µl of ligation to 50µl of cells, mix by stirring gently with tip & incubate on ice for 20 minHeat shock in water bath at 42°C for 30 secReturn tubes to ice for 2 minAdd 53µl of transformed cells to 250µl of LB and incubate at 37°C for 60 minMelt LB agar in microwave and keep at 50°C in water bathPrepare 50ml of LB+Agar (2 plates): Add 37.5µl Carbencillin (Stock: 100mg/ml Plate:75µg/ml), 50µl of X-Gal /IPTG (Stock: 2%/0.1M Plate: 1:1000)Plate 150µl of LB+cells onto 1 plate and incubate at 37°C over night
?. [Identify colonies (Day 3)]
Remove plates from 37°C the next morning and check for coloniesKeep in the fridge for a couple of hours or until next morning so blue/white colours become more intensePrepare master mix using a primer in the insert and the M13 fwd/rev primers to verify insert. (Can use blue colonies and M13 primers as neg. control)Prepare 50ml of LB+Agar (2 plates): Add 37.5µl Carbencillin (Stock: 100mg/ml Plate:75µg/ml), 50µl of X-Gal /IPTG (Stock: 2%/0.1M Plate:  1:1000) and pick verified colonies as stockGrow verified colonies over night in LB + Carbencillin (5ml LB + 5µl Cabencillin (100mg/ml dilutes to 100µg/ml))
?. [Wizard Plus Promega Mini Prep Kit (Day 4)]
Prepare a glycerol stock:Mix 150µl cells with 150µl glycerolStore at -80°C

Production of clear lysate:

Pellet 5ml of overnight culture for 5 minutes at 4000rpm.Thoroughly resuspend pellet with 250µl of Cell Resuspension Solution.Add 250µl of Cell Lysis Solution to each sample and leave until it clears up; invert 4 times to mix.Add 10µl of Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature.Add 350µl of Neutralization Solution; invert 4 times to mix.Centrifuge at top speed for 10 minutes at room temperature.

Binding of Plasmid DNA:

Insert Spin Column into Collection Tube.Decant cleared lysate into Spin Column.Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough, and reinsert Column into Collection Tube.

Washing:

Add 750µl of Wash Solution (ethanol added). Centrifuge at top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube.Repeat Step 1. with 250µl of Wash Solution.Centrifuge at top speed for 2 minutes at room temperature.

Elution:

Transfer Spin Column to a sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at top speed, then transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube.Add 100µl of Nuclease-Free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature.Discard column, and store DNA at –20°C or below.
?. [Send to sequencing (Day 4)]
Measure DNA concentration on plate reader and check 260/280 & 260/230 valuesDilute samples to 100ng/µlPrepare a 1:3 dilution (to 3.2pmol/µl) of sequencing primers