### PCR and TOPO TA Cloning for community analysis (7-13-05)

 

1.  PCR: Perform PCR reactions using the following primers.

# 1492R (Bacteria/Archaea-specific) 5'-GGTTACCTTGTTACGACTT-3'

# 27F (Bacteria-specific) 5'-AGAGTTTGATCCTGGCTCAG-3'

> **4Fa** (Archaea-specific) 5'- TCCGGTTGATCCTGCCRG-3'\*
>
> Typical base degeneracies have been left out except where indicated.\*
> Apparently, leaving them out results in better coverage of diversity
> (Ed DeLong).
>
> \* should retain the G/A (R) wobble since it is present in the 3' end
> but it could be substituted for a G only if G-T mismatch does not
> prevent primer binding
>
> **Bacterial primer set: 27F-1492R**
>
> **Archaeal primer set: 4Fa-1492R**
>
> a\. Typical reaction composition:

  ----------------- ------------------- ------------------- -------------------
  **Component**     **Concentration**   **Volume per        **Final
                                        reaction**          concentration**

  **Buffer          10X                 2.5                 1X
  w/MgCl2**                                                 

  **Forward         3µM                 2.5                 300 nM
  primer**                                                  

  **Reverse         3µM                 2.5                 300 nM
  primer**                                                  

  **BSA**           20mg/ml             1.25                1 µg/µl

  **dNTP mix**      2.5mM each          2                   200 µM each

  **ExTaq**         5U/µl               0.125               0.625 U

  **Template**      100-500 ng          Variable            n/a

  **Total                                                    
  Reagents**                                                

  **H~2~O**                             Balance              

  **Total volume**                      25                   
  ----------------- ------------------- ------------------- -------------------

> b\. Typical reaction conditions:

  ----------------------- -------------------- ---------------------------
  *95*                    *3 min*              

  **95**                  **30s**              **25 cycles^‡^**

  **48-58\***             **25s**              

  **72**                  **2min**             

  *72*                    *10 min*             

  *4*                     *storage*            
  ----------------------- -------------------- ---------------------------

> \* easiest to perform using a gradient cycler. Each sample should be
> amplified individually with 8 different annealing temps between 48-58°
> C (1 sample per well -- 8 wells per sample).
>
> **^‡^** if no product visible after 25 cycles can go up as far as 30
> cycles, but always try 25 first.

2.  Combine 8 annealing temp reactions for each sample and check results
    on a 2% gel. If multiple bands noted then GEL PURIFY your band of
    interest with a suitable kit. AVOID FREEZING PCR PRODUCT (freezing
    will remove some of the A overhangs and reduce ligation efficiency).

**CLONING NOTE**: If you freeze your PCR product, you may need to
replace some A overhangs to ensure adequate ligation efficiency into
TOPO TA. To do this, add Ex Taq polymerase buffer, 0.25 μl 10 mM dATP,
and 0.5 unit of *Taq* polymerase to purified PCR product. Incubate the
reaction for 10-15 minutes at 72°C (using a heatblock, waterbath or
thermocycler).  

3.  Prepare for the Transformation Reaction:

    a.  Set water bath to 42° C (check with thermometer it must be
        EXACTLY 42^o^ C).

    b.  Warm SOC medium (typically aliquoted \~ 250 μl in freezer) to
        room temperature.

    c.  Warm pre-poured LB agar plates containing 50 μg/ml ampicillin at
        37° for 30 minutes.

    d.  Thaw 1 vial of One Shot cells (**[on ice]{.underline}**) for
        each transformation.

4.  Set up Cloning Reaction. Aim for an insert to vector ratio of 3:1 to
    maximize diversity recovery. 1 μl of TOPO Vector (pCR4-TOPO)
    contains 10 ng DNA which is equivalent to 3.9 fmol DNA; therefore
    try to use 10-12 fmol 16S PCR product. That is equivalent to 9.9 --
    11.9 ng full length 16S rDNA PCR product.

  PCR product x µl (from Step 1)

> Salt Solution 1 µl (in kit in -20°C freezer)
>
> TOPO Vector 1 µl (in kit in -20°C freezer)
>
> [Sterile water balance]{.underline}
>
> FINAL VOLUME 6.00 µl

5.  Incubate. Gently mix by tapping the tube (DO NOT MIX BY PIPETTING)
    and incubate at room temperature for 30 minutes. After incubation,
    place the reaction on ice (or store overnight at -20°C).

6.  Transform into Competent Cells:

    a.  To one vial of One Shot Chemically Competent *E. coli* cells add
        2 μl of the cloning reaction (you can keep the rest at -80^o^C)
        and mix gently by tapping the tube. DO NOT MIX BY PIPETTING.

    b.  Incubate on ice for 5 minutes

    c.  Heat-shock the cells for 30 seconds at EXACTLY 42°C (in water
        filled heat/block or waterbath) without shaking.

    d.  Immediately transfer the tubes to ice.

    e.  Add 250 µl of the room-temperature SOC medium.

    f.  Incubate tubes (horizontally) for 1 hour in the 37° shaking
        incubator at 200 rpm.

    g.  Add 36 μl of sterile (autoclaved) 80% glycerol to achieve final
        conc. of 10% glycerol -- mix gently with pipette and transfer to
        cryo-tube.

    h.  Using sterile technique, spread 10 and 20 µl from each
        transformation onto 2 prewarmed LB plates to test
        transformation/ligation efficiency. Let LB plates stand
        right-side up for about 5 minutes to allow the cells to adhere
        to the agar, then invert and incubate overnight at 37°C. Place
        glycerol stocks in -80^o^C.

7.  Select and Analyze Colonies. Count colonies on each plate. Pick
    sample (24) of colonies with toothpick into PCR tube containing 5 μl
    sterile water and analyze colonies for inserts via PCR using M13
    primers.

> Cycling conditions:

  ----------------------- -------------------- ---------------------------
  *95*                    *10 min*             

  **95**                  **30s**              **30 cycles**

  **53**                  **30s**              

  **72**                  **1.5min**           

  *72*                    *7 min*              

  *4*                     *storage*            
  ----------------------- -------------------- ---------------------------

8.  Run the PCR products on a 2 % agarose gel to confirm bands of
    expected size and record percent of samples with correct inserts
    (should be 100% clones).

**CLONING NOTES:**

According to JGI, the most important parts of this procedure are to make
sure the final glycerol stock concentration is 10% (higher glycerol
adversely affects rolling circle amplification-RCA). The transformation
efficiency must be reported accurately and the QC PCR must yield 100%
clones with the correct insert size.

The type of *Taq* used is also important, it should have high
processivity to reduce stalling and chimera formation. ExTaq (Takara)
appears to be fine.

PCR®4-TOPO® allows direct selection of recombinants via disruption of
the lethal *E. coli* gene, *ccd*B (Bernard and Couturier, 1992; Bernard
*et al.*, 1994; Bernard *et al.*, 1993). The vector contains the *ccd*B
gene fused to the C-terminus of the LacZα fragment. Ligation of a PCR
product disrupts expression of the *lac*Zα-*ccd*B gene fusion permitting
growth of only positive recombinants upon transformation in TOP10 cells.
Cells that contain non-recombinant vector are killed upon plating.
**Therefore, blue/white screening is not required.**