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| - | ==Making LB Medium (P1.)== | + | =Protocols= |
| - | Luria-Bertani Medium (aka L-Broth or LB Medium). (Bertani says LB really stands for lysogeny broth.) LB is a standard growth medium for a variety of bacteria and conditions.
| + | ===Preparations=== |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Preparing_Media|Preparing media]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Making_Competent_Cells|Preparation of chemically competent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Making_electrocompetent_Cells|Preparation of electrocompetent cells]] |
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| - | ===Ingredients=== | + | ===Cloning=== |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Miniprepping|Miniprepping]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Digestion|Digestion]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Ligations|Ligations]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Transforming_Competent_Cells|Transforming chemically competent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/Transforming_electrocompetent_Cells|Transforming electrocompetent cells]] |
| | + | *[[Team:Amsterdam/Notebook/Protocols/ColonyPCR|Colony PCR]] |
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| - | #10 g Bacto-tryptone
| + | ===BioBrick characterization=== |
| - | #5 g yeast extract
| + | *[[Team:Amsterdam/Notebook/Protocols/Growth_Curve|Growth curve measurement]] |
| - | #10 g NaCl
| + | *[[Team:Amsterdam/Notebook/Protocols/Freeze_thaw|Freeze/thaw survival measurement]] |
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| - | ''Note:'' There are two formulations of LB, Miller and Lennox, that differ in the amount of NaCl. Lennox has less salt, only 5 g/L. The Qiagen miniprep kit recommends LB with 10 g NaCl for highest plasmid yields.
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| - | ===Protocol===
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| - | #Mix dry ingredients and add distilled water up to 1 Liter
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| - | #Pour into 2 L flask (or greater)
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| - | #Autoclave (liquid cycle)
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| - | #* 250°'''F''', 22psi, 30 minutes
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| - | Notes: We do not pH medium when we make it on the fly. However, if it is really important, pH the medium to 7.0 with 5M NaOH (~200µL). We usually obtain this from the kitchen.
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| - |
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| - | ===Source===
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| - | Adapted From:
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| - | J. Sambrook, D.W. Russell, ''Molecular Cloning: A Laboratory Manual'' (Cold Spring Harbor Laboratory Press, New York, ed. 3, 2001) pg. A2.2
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| - |
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| - | ==Making SOB Medium (P2.)==
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| - | SOB Medium. Used in growing bacteria for preparing chemically competent cells
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| - |
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| - | === Ingredients ===
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| - | *0.5% (w/v) yeast extract
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| - | *2% (w/v) tryptone
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| - | *10 mM NaCl
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| - | *2.5 mM KCl
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| - | *20 mM MgSO<sub>4</sub>
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| - | Per liter:
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| - | *5 g yeast extract
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| - | *20 g tryptone
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| - | *0.584 g NaCl
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| - | *0.186 g KCl
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| - | *2.4 g MgSO<sub>4</sub>
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| - |
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| - | ''Note:'' Some formulations of SOB use 10 mM MgCl<sub>2</sub> and 10 mM MgSO<sub>4</sub> instead of 20 mM MgSO<sub>4</sub>.
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| - | SOB medium is also available dry premixed from Difco, 0443-17.
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| - | Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.
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| - |
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| - | ===15/10 medium===
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| - | Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:
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| - | * 1.5% yeast extract
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| - | * 1% Bacto-Tryptone
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| - | * 10mM NaCl
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| - | * 2mM KCl
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| - | * 10 mM MgCl<sub>2</sub>
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| - | * 10 mM MgSO<sub>4</sub>
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| - |
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| - | ===Source===
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| - | Adapted From:
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| - | F. Ausubel et al., ''Short Protocols in Molecular Biology'' (John Wiley & Sons, ed. 4, 1999) pg. A1-36
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| - | ==Making SOC Medium (P3.)==
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| - | SOC Medium.
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| - |
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| - | ===Ingredients===
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| - | *[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]]
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| - | *20 mM glucose
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| - |
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| - | ===Protocol===
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| - | #Follow directions to make 1 liter of [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|Making SOB Medium (P2.)]] media
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| - | #After cooling medium to less than 50°C, add 20 ml filter sterilized 20% glucose solution
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| - |
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| - | ===Source===
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| - | Adapted From:
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| - | F. Ausubel et al., ''Short Protocols in Molecular Biology'' (John Wiley & Sons, ed. 4, 1999) pg. A1-36
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| - |
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| - | ==Making Competent Cells (P4.)==
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| - | This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [http://openwetware.org/images/b/bd/Pat6855494.pdf Bloom05 patent] as well. This protocol has been tested on NEB10, TOP10, MachI and [http://openwetware.org/wiki/Talk:TOP10_chemically_competent_cells BL21(DE3)] cells. See [http://openwetware.org/wiki/Bacterial_Transformation OWW Bacterial Transformation page] for a more general discussion of other techniques. The [http://openwetware.org/images/0/0c/Pat6960464.pdf Jesse '464 patent] describes using this buffer for DH5α cells. The [http://openwetware.org/images/c/c2/Pat6709852.pdf Bloom04] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.
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| - |
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| - | '''This is the chemical transformation protocol used by Tom Knight and the [http://partsregistry.org Registry of Standard Biological Parts].
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| - |
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| - | ===Materials===
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| - | *Detergent-free, sterile glassware and plasticware (see procedure)
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| - | *Table-top OD600nm spectrophotometer
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| - | *[[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]]
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| - |
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| - | ====CCMB80 buffer====
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| - | * 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
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| - | * 80 mM CaCl<sub>2</sub>.2H<sub>2</sub>O (11.8 g/L)
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| - | * 20 mM MnCl<sub>2</sub>.4H<sub>2</sub>O (4.0 g/L)
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| - | * 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/L)
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| - | * 10% glycerol (100 ml/L)
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| - | * adjust pH DOWN to 6.4 with 0.1N HCl if necessary
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| - | ** adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
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| - | * sterile filter and store at 4°C
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| - | * slight dark precipitate appears not to affect its function
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| - |
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| - | ===Procedure===
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| - | ====Preparing glassware and media====
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| - | =====Eliminating detergent=====
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| - | Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic
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| - | must be detergent free for these protocols. The easiest way to do this is to avoid washing
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| - | glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective
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| - | way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.
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| - |
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| - | =====Prechill plasticware and glassware=====
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| - | Prechill 250mL centrifuge tubes and screw cap tubes before use.
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| - |
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| - | ====Preparing seed stocks====
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| - | * Streak TOP10 cells on an [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] plate and grow for single colonies at 23°C
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| - | ** room temperature works well
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| - | * Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C
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| - | ** room temperature works well
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| - | * Add glycerol to 15%
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| - | * Aliquot 1 ml samples to Nunc cryotubes
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| - | * Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
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| - | ** This step may not be necessary
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| - | * Place in -80°C freezer indefinitely.
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| - |
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| - | ====Preparing competent cells====
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| - | * Inoculate 250 ml of [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P2.)|SOB]] medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3
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| - | ** This takes approximately 16 hours.
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| - | ** Controlling the temperature makes this a more reproducible process, but is not essential.
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| - | ** Room temperature will work. You can adjust this temperature somewhat to fit your schedule
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| - | ** Aim for lower, not higher OD if you can't hit this mark
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| - | * Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle.
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| - | ** Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
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| - | ** It is often easier to resuspend pellets by mixing ''before'' adding large amounts of buffer
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| - | * Gently resuspend in 80 ml of ice cold CCMB80 buffer
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| - | ** sometimes this is less than completely gentle. It still works.
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| - | * Incubate on ice 20 minutes
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| - | * Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
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| - | * Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
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| - | * Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
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| - | * Incubate on ice for 20 minutes
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| - | * Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
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| - | * Store at -80°C indefinitely.
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| - | ** Flash freezing does not appear to be necessary
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| - | * Test competence (see below)
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| - | * Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
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| - |
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| - | ====Measurement of competence====
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| - | * Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
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| - | ** This is at 10 pg/μl or 10<sup>-5</sup> μg/μl
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| - | ** This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
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| - | * Hold on ice 0.5 hours
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| - | * Heat shock 60 sec at 42C
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| - | * Add 250 μl [[Team:Amsterdam/Notebook/Protocols#Making SOB Medium (P3.)|SOC]]
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| - | * Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
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| - | ** using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
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| - | ** For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
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| - | ** Ampicillin and kanamycin appear to do fine with 1 hour growth
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| - | * Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
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| - | ** Good cells should yield around 100 - 400 colonies
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| - | ** Transformation efficiency is (dilution factor=15) x colony count x 10<sup>5</sup>/µgDNA
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| - | **We expect that the transformation efficiency should be between 5x10<sup>8</sup> and 5x10<sup>9</sup> cfu/µgDNA
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| - | ===5x Ligation Adjustment Buffer===
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| - | * Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
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| - | * KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)
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| - | * CaCl<sub>2</sub> 400 mM (200 ml/l of a 2 M solution)
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| - | * MnCl<sub>2</sub> 100 mM (100 ml/l of a 1 M solution)
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| - | * Glycerol 46.8% (468 ml/liter)
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| - | * pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter)
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| - | ** Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --[[User:Meagan|Meagan]] 15:50, 25 January 2007 (EST)
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| - | * water to 1 liter
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| - | * autoclave or sterile filter
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| - | * Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
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| - | *'''Reshma P. Shetty 10:49, 11 February 2008 (CST)''': Use of the ligation adjustment buffer is optional.
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| - |
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| - | ===References===
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| - | # Hanahan91 pmid=1943786
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| - | # Reusch86 pmid=3536850
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| - | # Addison04 pmid=15470891
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| - | # Bloom04 US Patent 6,709,852 [http://openwetware.org/images/c/c2/Pat6709852.pdf pat6709852.pdf]
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| - | # Bloom05 US Patent 6,855,494 [http://openwetware.org/images/b/bd/Pat6855494.pdf pat6855494.pdf]
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| - | # Jesse05 US Patent 6,960,464 [http://openwetware.org/images/0/0c/Pat6960464.pdf pat6960464.pdf]
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| | {{:Team:Amsterdam/Footer}} | | {{:Team:Amsterdam/Footer}} |
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