ribose cloning experiment
ribose cloning experiment
This scheme is just one way of treating PCR products with RNAase. There are many alternative and effective ways of purifying the PCR product to remove the primers, then treating the PCR product with the enzyme, and then removing the enzyme. It is important to note that it is easy to set up a temperature bath program with a PCR instrument. Optionally, a 'cooling' step can be added after the heating step, i.e. 5 min or even overnight in the cold module. This experiment is based on the PCR Laboratory Guide (2nd edition) by Kang Seed and Lijia Qu.
Operation method
ribose cloning experiment
Materials and Instruments
ATEN buffer DNA buffer (TEN) KLA buffer T5E5 Dpn I Klentaq LA Proteinase K Proteinase K storage buffer RNAase A Designed vectors Primers that have been biotin-TEG-modified at the 5' end and modified at the 3' end with ribosylcytosine or ribosyluracil Kanamycin (Sigma) Tetracycline Ticarcillin Move -Materials For more product details, please visit Aladdin Scientific website.
Electrotransformation apparatus PCR (thin-walled) tubes tubes with regular wall thickness Streptomyces anti-biotin protein beads
1. Buffers, solutions and reagents .
10XATEN buffer .
0.5mol/LTris-HCl,pH7.9
2.5mol/L sodium chloride
0.25mol/L Na4EDTA,pH7.9
Betaine (SigmaB-2629)
Blue dextran (Sigma D-5751)
DNA buffer (TEN)
10mmoI/LTris-HCl,pH7.9
10mmol/L NaCl
0.1mmol/L EDTA
Ethanol, 75% with 25% DNA buffer
10X KLA buffer
500 mmol/L Tris-HCl base (Sigma; TrizmaBase)
160 mmol/L Ammonium Sulfate
1% Tween20
25 mmol/L Ferric Chloride
Adjust pH to 7.9 by adding hydrochloric acid drop by drop to a pH of 9.2 before adjustment. Remove droplet individually to check pH to prevent contamination of the buffer by DNA on the pH meter probe.
30% polyethylene glycol 3350 (PEG), 1.5 mol/L NaCl
Sodium acetate, 3 mol/L, pH 5.6
T5E5
5 mmol/L Tris-HCl, pH 7.9
5 mmol/L Na4EDTA,pH7.9
2. Enzyme and enzyme buffer
Dpn I
Klentaq LA (Barnes 1994; Clontech 8421-1)
Protease K (Roche1373196)
Protease K Storage Buffer
100umol/L β-mercaptoethanol
20 mmol/L Tris-HCl, pH 7.9
1 mmol/LCaCl2
50% (v/v) glycerol (630 g/L)
RNAase A (Ambion2270 or 2272)
3. Nucleic acids and oligonucleotides
Designed vectors
Primers that have been biotin-TEG-modified at the 5' end and ribosylcytosine or ribosyluracil-modified at the 3' end.
4. antibiotic
Kanamycin (Sigma), final concentration 25ug/ml.
Tetracycline (Calbiochem) at a final concentration of 12ug/ml.
Ticarcillin (SmithKline Beecham), final concentration 100ug/ml
5. Specialized equipment
Electroconversion apparatus
PCR (thin-walled) tubes; tubes with regular wall thickness; 0.5 ml or 1.7 ml
Streptavidin anti-biotin protein beads
6. Cells and tissues
Electroshock receptor cells (Dower et al. 1988; Invitrogen 18290-015)
II. METHODS
1. Target fragments and vector sequences were amplified using primers terminating in ribosylcytosine or ribosyluracil and KLA buffer (pH 7.9) in 1.3 mol/L and 1.9 mol/L of betaine, respectively. The plasmid replicons of some vectors amplified better in the standard long PCR reaction at pH 9.2, but the colEl replicons amplified well at pH 7.9.
For optimal amplification efficiency, primers that have been biotin-TEG-modified at the 5' end and streptavidin-resistant beads, as described below, should be used. Klemaq LA enzyme mixture (BameS 1994) is more productive than other DNA polymerases, and it is more resistant to betaine (Baskaran et al. 1996).
PCR using the plasmid as a template is slightly more efficient and the final background caused by transformation of the template into E. coli cells is lower if the template is linearized with a restriction enzyme prior to PCR.
If the template used to amplify the construct or target fragment is from dam+ E. coli, Dpn I selection is usually a good approach and may result in unwanted background in subsequent cloning steps. dpn I cannot digest the PCR product because it contains unmethylated GATC sites (Weiner et al. 1994).
2. Add 1ul (10U) of Dpn I to each 100ul of PCR reaction and incubate at 37°C for 2 h. The PCR product should not be digested with Dpn I because it contains a non-methylated GATC site (Weiner et al. 1994).
This treatment reduces the background of vector alone or target fragment alone to 0-20 colonies.
3. PEG precipitation step. The purpose of this step is to remove primers and salts. Transfer the PCR reaction to a thick-walled (uniform wall thickness) tube, either 0.5 ml or 1.7 ml, as thin-walled PCR tubes cannot be used for centrifugation. Then, add 5ul of lmg/ml blue dextran (to show the location of the precipitate) and 1/2 volume of 30% PEG3350, 1.5 mol/L NaCl (eventually 10% PEG, 0.5 mol/L NaCl). Leave at room temperature for 30 min or overnight at 4°C. Centrifuge for 15 min. Centrifuge for 15 min and pour off the supernatant looking at the blue precipitate to avoid accidentally pouring off the precipitate. If the precipitate is very thin and invisible, try to avoid pouring the precipitate, but keep the supernatant. Rinse the precipitate with 75% ethanol and centrifuge for 8 min. dry the precipitate in air for 10-20 min. resuspend the precipitate with 100ul of T5E5 buffer and place on ice for at least lOmin, with occasional shaking.
4. Pancreatic RNAase A step. Dilute 200ug of RNAase A with T5E5 buffer to 200ug/ml. DNA buffer can also be used (E. Oates, personal communication). Add 6ug (30ul) of RNAase to each 100ul of PCR product, shake and gently centrifuge. The RNAase-containing PCR product is then placed in a warm bath at 55°C for 30 min and cooled.
5. Proteinase K procedure. The enzyme should be stored at -20°C in Proteinase K Preservation Buffer (see material sheet). Add 5ug of Proteinase K or approximately the same mass (6ug) of RNAase as above, mix thoroughly, centrifuge briefly, warm bath at 65°C for 30 min, and cool.
If biotin primers are used, proceed immediately to the bead purification step below. Otherwise, the DNA is ready.
6. Cloning step. Take a 2-8ul sample of the vector or target fragment for agarose gel electrophoresis and place a concentration standard next to it to determine the concentration of both the vector DNA and the target fragment. They should be cloned in equal amounts, and it is not important whether the concentration is exact or not.
1~10ul Vector = 30~100ng, 0.02pmol
1~10ul Target fragment = 5~25ng (equal amount of target and vector substances)
Add 1XATEN buffer to 40ul
7. Take 20ul of each clone mixture for different controls, e.g., before heating, without heating, gel samples, etc. Proportion more of the desired clone mixture. Minimally use 40ul:20ul for gel electrophoresis and 20ul for cell transformation. 
8. Bead-purified DNA may not require annealing as it appears to self-assemble at room temperature (data not given), but this heating step is very helpful for non-biotin primers.
Bath at 78°C for 2 min, then 52°C for 30 min; alternatively, bath at 78°C for 2 min, then 65°C for lOmin, then cool slowly to 52°C or lower over >30 min. Save 20ul for use as a post-annealing gel test sample.
These gel samples can be used to detect and improve recombination efficiency. This step can be skipped for routine cloning, but, as with any DNA cloning experiment, if something goes wrong, the experimenter will not know what went wrong without gel analysis.
9. Add 1/9 or 1/6 volume of 3mol/L sodium acetate (pH 5.6) and 2 mg of blue dextran, precipitate the DNA by adding 2 to 3 times the volume of ethanol, refrigerate at -20°C for at least 30 min, and then centrifuge for 10 to 15 min. Wash the visible precipitate with 75% ethanol:25% DNA buffer, and then centrifuge for 8 min. In this 75% ethanol wash, a small amount of salt is added to the DNA buffer so that the 25bp sticky ends do not unravel in the following steps.
10. Resuspend the dried precipitate on ice with 22ul of water, the added water will turn a slight blue color due to the blue dextran.
Remove for use as an electrophoresis sample or as a backup for transformation.
11. Transform 70ul of S. galvanicus sensors with 15ul of DNA, add 1ml of enriched medium within 5s, and apply to appropriate antibiotic medium: 100ug/ml ticarcillin plates after 10min, or 25ug/ml kanamycin plates after 5-16 h at 30°C, or 12ug/ml tetracycline plates after 2 hours at 30°C. 12ug/ml tetracycline plates after 2 h at 30°C. Prolonged incubation time after electrotransformation is necessary to express resistance to some antibiotic genes.