Cloning PCR fragments

PCR purification with Ethanol precipitation

1. Add 2 volume 100% ethanol (usually about 40ul)
2. Mix and incubate for 15 min at RT
3. Centrifuge for 15 min at maximum speed
4. Discard ethanol and wash with 100 ul of 70% ethanol. Centrifuge 10 min at max speed
5. Discard ethanol and dry the pellet at room temperature (about 5 min)
6. Resuspend DNA in 20-40 ul ultra pure water depending on band intensity.
7. Run 2 uL of sample on 1% agarose gel to check quality
8. Measure DNA quantity on spectrophotometer (2 ul sample + 98 H2O)
9. Proceed to ligation 

QIAGEN PCR Cloningplus Kit Ligation Protocol 

Table 1. Guide for the amount of PCR product to use in the ligation reaction

* Calculated for 50 ng pDrive Cloning Vector using the following equation:  ng PCR product required = 50 ng x PCR product size (bp) x molar ratio 

Ligaion Procedure

1. Thaw 2x Ligation Master Mix, pDrive Cloning Vector DNA, and distilled water (provided). Place on ice after thawing. 

   It is important to mix the solutions completely before use to avoid localized concentrations of salts. Keep 2x Ligation Master Mix on ice and immediately store at –20°C or –70°C after use. 

2. Prepare a ligation-reaction mixture according to the following scheme:

    

   Component                                                Volume/reaction

   pDrive Cloning Vector (50 ng/μl)                          1 μl

   PCR product                                                         1–4 μl*

   Distilled water                                                      variable

   Ligation Master Mix, 2x†                                        5 μl

   Total volume                                                         10 μl

   * Purified PCR product. If using non-purified PCR product, do not add more than 2 μl PCR product.

   † We recommend adding the Ligation Master Mix last.

3. Briefly mix the ligation-reaction mixture then incubate for 30 min at 4–16°C (e.g., in a refrigerator, water bath, or thermal cycling block). Mix gently, for example by pipetting the ligation-reaction mixture up and down a  few times.

   Note: Increasing the ligation time to 2 h can result in a 2–3 fold increase of recombinants. This might be especially useful for PCR fragments longer than 2 kb. If the total number of recombinants is not essential, however, the ligation time can be as short as 15 min.

4. Proceed with the transformation protocol (page 14) or store ligation-reaction mixture   at –20°C until use. 

   IMPORTANT: The transformation protocol on page 14 is for use with QIAGEN EZ Competent Cells. If electrocompetent cells will be used, we strongly recommend inactivating the ligase in the ligation-reaction mixture prior to electroporation.

   Incubate the ligation-reaction mixture for 10 min at 70°C, then proceed with electroporation. Alternatively, the MinElute Reaction Cleanup Kit can be used to remove ligase from the ligation-reaction mixture. The ligase does not need to be inactivated when using QIAGEN EZ Competent Cells. 

QIAGEN PCR Cloningplus Kit Transformation Protocol

Important notes before starting:

• This protocol is for use with QIAGEN EZ Competent Cells. It is not for use with electrocompetent cells. If electrocompetent cells will be used, we strongly recommend inactivating the ligase in the ligation-reaction mixture prior to electroporation. See step 4 of the ligation protocol (page 13) for details.
• Competent cells are extremely sensitive to temperature and mechanical stress. Do not allow QIAGEN EZ Competent Cells to thaw at any point prior to transformation. Keep thawed cells on ice. Avoid excessive and/or rough handling, especially pipetting. Mix cells by gentle flicking.
• Thaw SOC medium and warm to room temperature. Store at –20°C or –70°C after use.
• Prepare fresh LB agar plates containing either ampicillin (100 μg/ml LB agar) or kanamycin (30 μg/ml LB agar) as a selection marker. Include IPTG (50 μM) and X-gal (80 μg/ml) for blue/white screening of recombinant colonies. See Appendix(page 27) for recipes. 

Procedure

1. Thaw the appropriate number of tubes of QIAGEN EZ Competent Cells on ice. Thaw SOC medium and warm to room temperature. 

   IMPORTANT: Competent cells should only be thawed on ice. Do not allow unused QIAGEN EZ Competent Cells to thaw. Test whether cells are thawed by gently flicking the tube. Proceed immediately to the transformation step once the cells have thawed.

2. Add 1–2 μl ligation-reaction mixture per tube of QIAGEN EZ Competent Cells, mix gently, and incubate on ice for 5 min.Mix gently, for example by flicking the transformation mixture a few times.
3. Heat the tube(s) in a 42°C water bath or heating block for 30 s without shaking.
4. Incubate the tube(s) on ice for 2 min.
5. Add 250 μl room temperature SOC medium per tube and directly plate 100 μl each transformation mixture onto LB agar plates containing ampicillin. 

   Note: For kanamycin selection, incubate the cells at 37°C for 30 min with shaking prior to plating to allow recombinant outgrowth.

   The transformation mixture can be plated using a sterile bent glass rod or a specialized spreader. It is generally recommended to plate different amounts of each transformation mixture onto separate plates (e.g. 100 μl and 20 μl) to ensure good separation of colonies for subsequent single-colony isolation. For more efficient plating of small volumes of transformation mixture (<50 μl) we recommend pipetting 100 μl LB medium onto the plate, and then pipetting the transformation mixture into the liquid LB.

6. Incubate the plate at room temperature until the transformation mixture has absorbed into the agar. Invert the plate and incubate at 37°C overnight (e.g., 15–18 h). 

   Note: For blue/white screening, we recommend a second incubation at 4°C (e.g., in a refrigerator) for a few hours. This “cold” incubation step enhances blue color development and thereby facilitates differentiation between blue colonies and white colonies. 

Colony PCR

Date of experiment:

Operator:

Samples: 

Protocol for lysing bacterial colony

1. Pick a colony from agar plate using a 200ul pipet tip or sterile toothpick.
2. Transfer the bacteria to a 0.5ml tube containing 50ul of sterile water. Vortex to disperse the pellet. *If you need to keep the colony, transfer 10ul of suspended bacteria to 100ul LB
3. Place the tubes in boiling water or a heat block or Thermocycler (PE 480) at 99C for 5 minutes to lyse the cells and denature DNases.
4. Centrifuge at 12,000 x g for 1 min to remove cell debris.
5. Transfer 10ul of the supernatant to a fresh 0.5ml tube for PCR. Leave on ice until use or store in refrigirator. 

PCR set up with universal primers (T7 and SP6)