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"Microsatellites" Home | |  |   |

In our project, we started with existing primers to generate PCR products for analysis.  Because it is difficult to identify microsatellite regions in a genome, a classroom project should begin by identifying collectible species for which primers are available.   If primers are not available, it is necessary to prepare a genomic library which is screened for microsatellite regions by sequencing cloned inserts.  This process requires doing a genomic digest, inserting the restriction fragments into plasmids, transforming and plating out bacteria to amplify and isolate clones containing the inserts.  The protocols for these procedures follow.

Restriction Enzyme Digest Protocol

1. Samples of DNA from those extracted were mixed with restriction enzyme XbaI in a graduated series of volumes.  The following solutions were mixed for these samples, in the order given (all volumes are in microliters):

2. The samples were incubated at 37 degrees C for 8 hours or overnight.

3. Load 1 microliter of dye and 7 microliters of digest sample on an E-gel and run the gel for 30 minutes to verify that genomic DNA is present.  Genomic DNA will have all sizes of fragments and therefore a smear will appear, rather than bands of distinct sizes.

Purification of Restriction Digest Protocol

1. Restriction digested samples were purified using the Qiaquick Purification Kit according to the following procedure.

2. 500 microliters of Buffer PB were added to 100 microliters of restriction digest PCR product.

3. The 600 microliters resulting were transferred into the center of a Qiaquick spin column over a collection tube and centrifuged for 30-60 seconds.

4. The liquid was discarded, the spin column returned to the same collection tube and 750 microliters of Buffer PE used to wash the sample.  Centrifuge for 30-60 seconds.

5. The liquid was discarded, the spin column returned to the collection tube and the column centrifuged for an additional 1 minute at full speed.  The collection tube was then discarded.

6. The spin column was placed over a clean 1.5 ml tube and 20 microliters of Buffer EB (containing 10 mM Tris-Cl at pH 8.5) added to the center of the column.  The column was allowed to stand for 1 minute at room temperature, then centrifuged for 1 minute.

TOPO Cloning Protocol

Step 1.  Preparation of bacterial plates

1. Prior to beginning the process of cloning, agar plates must be prepared which can be done using a packet of dry Invitrogen imMedia Amp Blue medium.

2. b. The dry medium was dissolved in 200 ml of distilled water, microwaved on medium power for 3-4 minutes until completely dissolved and cooled slightly until it can be handled. 

3. c.  It was poured into 10 pre-sterilized plates, leaving the lid open to prevent condensation during cooling.

Step 2.  Addition of 3' A-overhangs

1. 3' A-overhangs were added to the samples in order to take advantage of the TOPO plasmid vector which was a linearized vector ending in single 3' T-overhangs.  This process works for blunt-end inserts as well as inserts digested by several different restriction enzymes. 

2. A mixture of 2.5 microliters of Taq buffer, 2.5 microliters of dNTP, and 0.1 microliters of Taq was prepared to which 20 microliters of the DNA digest was added. 

3. The resulting mixture was placed on a heat block at 72 degrees C for 10 minutes to attach the 3' A-overhang to the insert. 

4. The samples were then kept on ice until ready to be used.

Step 3.  Set up for Cloning

1. To increase the number of transformants that result from the cloning reaction, a salt of NaCl/MgCl₂ was added, by mixing 1 microliter of salt solution (included in the kit) with 2 microliters sterile distilled H₂O.

2. This was added to 2 microliters of digested and purified DNA, followed by 1 microliter of TOPO vector. 

3. The tubes were incubated for 5 minutes at room temperature and then placed on ice.

Step 4.  Transformation into Competent E. coli Cells

1. A water bath was brought to 42 degrees C. 

2. From each of the samples in step 3, 2 microliters of TOPO vector/DNA digest were added to 1 vial of Invitrogen One Shot Chemically Competent cells.

3. This was mixed gently but NOT by pipetting up and down.

4. The samples were incubated on ice for 15 minutes, after which they were heat shocked for 30 seconds at 42 degrees C EXACTLY!  (Time and temperature are both crucial.)

5. Samples were transferred immediately back to the ice bath.

6. Room temperature SOC medium in the amount of 250 microliters was added to the samples.  The tubes were capped tightly and placed in a shaking water bath at 37 degrees C for 45 minutes.

7. Aliquots of 25 microliters, 50 microliters and 100 microliters were spread on the LB bacterial plates.  Spreading was done with a glass spreader, dipped into a petri plate containing ethanol (ETOH) and burned off in a flame.  The spreader was cooled by running it over the under surface of the lid of the bacterial plate and then the bacteria spread thoroughly over the surface of the plate. 

8. The plates were incubated at 37 degrees C for 12 - 16 hours or overnight.  Lab surfaces were cleaned with a small amount of alcohol.

9. The linearized vector includes both the lacZ gene as well as an ampicillin-resistance gene.  When the lacZ gene is intact, a component of the LB medium activates it and the enzyme produced will digest X-gal (another component of the LB medium), producing a bright blue compound.  If the lacZ gene sequence is disrupted by the transformant (the DNA insert), it will not produce the enzyme because of a frameshift and will not digest X-gal.  Therefore, bacteria that do NOT contain the DNA insert will have intact lacZ genes and will produce bright blue colonies.  Those that contain the DNA insert will disrupt the gene and produce white colonies.

10. Additional discussion of the process of cloning with a protocol similar to that for the TOPO cloning vector is included in paragraphs 3 and 4 of an article on Microsatellite DNA.

11. For information on designing primers for microsatellites, see Primer Design.