Protocol

E. coli Calcium Chloride competent cell protocol

   1. Inoculate a single colony into 5mL Lb in 50mL falcon tube. Grow O/N @ 37°C.

   2. Use 1mL to inoculate 100mL of LB in 250mL bottle the next morning.

   3. Shake @ 37°C for 1.5-3hrs.

Or

   1. Inoculate a single colony into 25mL LB in a 250 mL bottle in the morning.

   2. Shake @ 37°C for 4-6 hrs.

Then….

   1. Put the cells on ice for 10 mins (keep cold form now on).

   2. Collect the cells by centrifugation in the big centrifugue for 3 mins @ 6000rpm

 3. Decant supernatant and gently resuspend on 10 mL cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).

   4. Incubate on ice x 20 mins

   5. Centrifuge as in 2

   6. Discard supernatant and gently resuspend on 5mL cold 0.1MCaCl/15%Glycerol

   7. Dispense in microtubes (300μL/tube). Freeze in -80°C.

Transformation of Ca++ competent cells

   1. Put 1μL of circular plasmid or all of a ligation reaction of plasmid DNA in a microtube. Gently add ~100μL of competent cells. Do NO DNA control tube with cells and no DNA.

   2. Incubate for 30 mins on ice.

   3. Heat shock for 2 mins @ 42°C. Put back on ice.

   4. Add 900 μL of LB to tubes. Incubate @ 37°C for 30 mins.

   5. Plate 100-1000 μL of the cells in LBAmp or LBCarb (100µg/ml) plates. Plate 100 μL of the NO DNA control in a blood plate (to check for quality of cells). Grow O/N. U can save the rest in the cold room or freeze with 15% of Gly in case u get no colonies (v. unlikely).

   6. If you need a lot of colonies or the ligation is of low efficiency, centrifuge the transformation for 1 min @ 8000 rpm, discard 900 μL of supernatant, resuspend on the 100 μL left and plate the whole lot.

Ethanol precipitation of small fragments

   1. Add 2 volumes ice cold absolute ethanol to sample. Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.

   2. Incubate 1 hr at -80°C. The long incubation time is critical for small fragments.

   3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).

   4. Remove supernatant.

   5. Wash with 750-1000 μL room-temperature 95% ethanol. Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.

   6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).

   7. Let air dry on benchtop. I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.

   8. Resuspend in an appropriate volume of H2O. Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H2O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H2O has the advantage of keeping the salt content of your ligation reaction down.

Restriction Digestion

Ligation Reaction

3A Assembly

   1. Miniprep your two parts.

   2. Digest your two parts and construction plasmid backbonedestination vector with the following enzymes

     ❧ Left part with EcoRI and SpeI

     ❧ Right part with XbaI and PstI

     ❧ Construction plasmid backbone with EcoRI and PstI. Also digest the construction plasmid backbone with DpnI if possible to eliminate any plasmid remaining from the PCR.

   3. Set the ligation reaction.

   4. Transform the ligation product. (See Chemical transformation or Electroporation.)

   5. If the input parts are good, almost all colonies will be correct.

   6. If desired analyze the transformation with single colony PCR followed by agarose gel electrophoresis.

     ❧ In rolling, large scale assembly, this step is often omitted.

   7. Miniprep clones that generated a band of the appropriate size. 8. Sequence the clone. 9. Record the sequence information in the Registry.

Isoproponal Precipitation

   1. Adjust the salt concentration with sodium acetate (0.3 M, pH 5.2, final concentration).

   2. Add 1 volumes of room-temperature isopropanol to the DNA solution and mix well.

   3. Centrifuge the sample immediately at 10,000–15,000 x g for 30 min at 4°C

   4. Carefully decant the supernatant without disturbing the pellet.

   5. Wash the DNA pellet by adding 1 ml (depending on the size of the preparation) of room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

   6. Centrifuge at 15,000 x g for 10 min at 4°C.

   7. Carefully decant the supernatant without disturbing the pellet.

   8. Air-dry the pellet for 5–20 min (depending on the size of the pellet).

   9. Redissolve the DNA in a suitable buffer.

Tip: Use a buffer with a pH of 7.5–8.0, as DNA does not dissolve easily in acidic buffers. Often distilled water can have an acidic pH. The addition of EDTA protects the DNA from DNase digestion.