I. PCR SELECT Spin Column Chromatography Protocol
Please read through the entire protocol before proceeding.
The PCR SELECT-III spin columns are intended for use in removing unincorporated deoxynucleoside triphosphates (dNTPs), residual mineral oil, polymerase, primers, primer-dimers from PCR reactions. To maximize recoveries, this column should be used to purify double-stranded PCR products greater than 300 base pairs (bp) in length. After brief centrifugation in a swinging bucket or horizontal rotor, the purified nucleic acid is recovered from the column without significant change in volume.
Note: If the 300 bp "cut-off" of the PCR SELECT-III spin columns is not suitable, alternative spin columns are available separately from 5 Prime -> 3 Prime (see table on page 5). PCR SELECT-I columns are intended for use in removing unincorporated dNTPs, residual mineral oil, polymerase, and primers with maximal recovery of PCR products greater than approximately 20 bp. PCR SELECT-II columns are intended for use in removing unincorporated dNTPs, residual mineral oil, polymerase, primers, and primer-dimers with maximal recovery of PCR products greater than approximately 100 bp. Alternatively, the PRIME PCR CLONER is available with PCR SELECT-II columns (Catalog No: 1-543210).
Spin Chromatography Procedure:
1. Invert column several times to fully resuspend the gel. Remove top closure (large) first and then bottom closure (small).
2. Place column in one of the collection tubes provided and centrifuge at 1000 xg (rmax) for 1 minute at speed in a centrifuge with a swinging bucket or horizontal rotor. A larger centrifuge tube may be used as a carrier for the column/collection tube assembly.
3. Empty collection tube of the collected buffer and place the column back into the same collection tube.
4. Centrifuge at 1000 xg (rmax) for 2.5 minutes at speed.
5. Discard collection tube and collected buffer.
6. Place the column in a second collection tube, carefully apply the 50-100 uL PCR sample directly to the center of the shrunken gel bed, and centrifuge at 1000 xg (rmax) for 2.5 minutes at speed.
7. The PCR product will be recovered in the collection tube in TE, pH 8 at approximately the same volume that was added to the column.
8. The concentration of PCR product should be estimated by agarose gel electrophoresis or abosorbance.
As greater than 99% of the unincorporated dNTPs, primers, and short primer-dimers will be retained in the column gel, discard the used column in an appropriate fashion. NOTE: The PCR SELECT spin columns are intended for one PCR product purification only.
II. Prime PCR Cloner Cloning Protocol
Please read through the entire protocol before proceeding.
NOTE: Please briefly centrifuge system tubes before opening to consolidate reagents.
NOTE: All values given are based on a 20 uL ligation reaction volume. Although a wide range of PCR product concentrations can be used in the cloning and subsequent ligation reactions, optimal cloning efficiencies occur when the amount of PCR prduct added to the ligation reaction (Section III below) is in the range of a 2:1 to 8:1 molar ration of PCR product to pNoTA/T7 vector. The appropriate amount of PCR product to add to the PRIME PCR cloning reaction can be calculated from the following table:
|
Effective |
PCR Product |
Multiplication |
ng of PCR |
|
0.5:1 |
0.056 |
||
|
1:1 |
0.111 |
||
|
2:1 |
0.222 |
||
|
4:1 |
0.444 |
||
|
8:1 |
0.889 |
Cloning Procedure:
NOTE: Steps 1 through 4 below must be performed on ice.
1. Prpare for two incubations, one at 16 C and another at 75 C. The thermal cycler can be used for these incubations.
2. Using the table above, calculate the amount and corresponding volume of column-purified PCR product (Section I, step 7) to use in the PRIME PCR cloning reaction.
3. In a 0.5-0.65 mL microcentrifuge tube on ice, combine:
|
Volume |
Reagent |
|
uL |
Molecular Biology Grade Water |
|
2 uL |
10X PRIME PCR Cloning Reagent |
|
1 uL |
PRIME PCR CLONER Nucleotide Stock |
|
1 uL |
0.1 M DTT Solution |
|
uL |
Column-processed PCR Product from Step 2 Above |
|
18.5 uL |
Combined Reaction Volume |
NOTE: With the initial use of the PRIME PCR CLONER, we recommend that a separate control reaction be performed to test the overall efficiency. In a separate 0.5-0.65 mL microcentrifuge tube, combine:
|
Volume |
Reagent |
|
13.5 uL |
Molecular Biology Grade Water |
|
2 uL |
10X PRIME PCR Cloning Reagent |
|
1 uL |
PRIME PCR CLONER Nucleotide Stock |
|
1 uL |
0.1 M DTT Solution |
|
1 uL |
System Test DNA |
|
18.5 uL |
Combined Reaction Volume |
[Following transformation, system test DNA should yield greater than 500 bacterial colonies per plate with 50% or more being white.]
4. Mix the combined reaction volume briefly and add 1.5 uL PRIME PCR Modification Reagent to each tube.
5. Mix well by gentle vortexing, centrifuge briefly at 4 C to consolidate contents to the tube bottom, and incubate at 16 C for 15 minutes.
6. Heat the cloning reaction at 75 C for 15 minutes. During that time, please review the protocol and prepare for the ligation below.
7. After the heating step is completed, set the tube at room temerature until it is needed in the ligation protocol. (Note: It is not necessary to centrifuge the tube after heating.)
III. Prime Efficiency DNA Ligation Protocol
Please read through the entire protocol before proceeding.
1. Prepare for two incubations, one at 25 C and another at 65 C. The thermal cycler can be used for these incubations.
2. In a fresh 0.5-0.65 mL microcentrifuge tube at room temperature, combine:
|
Volume |
Reagent |
|
5 uL |
Molecular Biology Grade Water |
|
1 uL |
0.1 M DTT Solution |
|
2 uL |
10X PRIME Efficiency Ligation Buffer |
|
10 uL |
Cloning Reaction (from step 7 above; |
|
1 uL |
pNoTA/T7 vector DNA* |
|
19 uL |
Combined Reaction Volume |
3. Mix briefly and add 1 uL T4 DNA Ligase to each ligation reaction tube.
4. Mix well by gentle vortexing and centrifuge at room temperature to consolidate the contents to the tube bottom.
5. Incubate the ligation reaction at 25 C for 30 minutes. During that time, please review the protocol and prepare for the bacterial transformation below.
6. Following the 25 C incubation, heat the ligation reaction at 65 C for 2 minutes. This heating step must be performed for maximum efficiency in the subsequent transformation.
7. After the heating step is completed, centrifuge tube briefly to consolidate contents and then set the tube at room temperature until it is needed in the transformation protocol.
IV. Bacterial Transformation Protocol
Please read through the entire protocol before proceeding.
NOTE: The agar plates, with an appropriate antibiotic (ampicillin for pNoTA/T7), for the transformation plating below should be dried at room temperature for 30-45 minutes prior to spreading (steps 5 and 6 below).
1. Each transformation requires 200 uL of competent E. coli cells (one tube). Just prior to use, the appropriate number of competent cell tubes should be thawed on ice and the thawed tube(s) then kept on ice until used.
Alternatively, other competent E. coli bacterial strains capable of a-complementation (blue-white screening), e.g. JM 109, can be used for transformation. Please note that the bacterial competency should be at least 1 x 10^6 cfu per ug DNA.
2. Add all of the ligation reaction (step 7, Section III above) directly into 200 uL of competent cells. Mix gently by tapping the tube. A positive control transformation containing 1 uL (1 ng) of pUC 18 Transformation Control DNA (provided) in 19 uL water should be included. We recommend that a control transformation lacking DNA be included as well.
3. After mixing the DNA with the cells, incubate the tube on ice for 20 minutes.
NOTE: Prepare the X-gal Solution by adding 1 mL of dimethylformamide to the tube containing the 20 mg of X-gal powder. Mix well and use. Store the remaining solution at -20 C protected from light.
4. Heat shock for 90 seconds at 42 C, and place the cells on ice.
5. To the center of one "well-dried" plate (containing 100 ug/mL ampicillin), pipet in order, 100 uL IPTG Solution, 40 uL X-gal Solution, and all 200 uL of the transformation reaction (from step 4 above).
6. Spread all of the pipetted material evenly across the plate and proceed to processing the next sample.
Alternatively, you may want to incorporate a recovery phase. In this case, the ligation reaction and competent cells are combined in a 1.5 mL or larger sterile centrifuge tube (Section IV, Step 2 above). After heat shocking, add 0.8 mL 2X YT, LB, or SOC broth to the transformed cells and incubate for 45-60 minutes at 37 C with shaking. Briefly centrifuge to pellet the cells, remove 800 uL of supernatant, resuspend cells into remaining supernatant, and spread all of the suspension onto a plate as in steps 5 and 6 above.
7. Incubate plates overnight at 37 C.
8. Following the incubation, white colonies of 0.5-1.0 mm in diameter and some smaller blue colonies (<0.1 mm diameter) should be evident. Select only the white colonies for analysis, preferably those near the center of the plate (to avoid potential false positives resulting from uneven distribution of X-gal and IPTG).
NOTE: On occasion, especially after prolonged incubation at 37 C, extremely small colonies may be seen on the plates. These background colonies do not have insert and should not be analyzed. Analyze only white colonies, which will be much larger in size.
9. For miniprep DNA preparation, we recommend that individual colonies be cultured in 2 mL of liquid medium (e.g. 2X YT, LB, etc. supplemented with 100 ug/mL ampicillin) overnight at 37 C with vigorous agitation.
10. Prepare plasmid DNA by our 2 minute miniprep procedure, INSTA-MINIPREP, by our non-organic PERFECT-prep method, or by alkaline lysis.
11. Analyze for insert by restriction enzyme digestion or other suitable procedures.
NOTE: Depending upon the quality and quantity of the PCR product or blunt-ended DNA fragment, the expected number of white colonies should range between 30 and 300 per plate. The number of blue colonies will also vary, decreasing with increasing PCR product to pNoTA/T7 vector molar ratio.