AT Cloning Kit with Competent Cells, 20 reactions
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BP-ATTMCloning Kit
For cloning PCR product generated with Taq DNA polymerase
Cat. No. PKC-201 (without competent cells)
Cat. No. PKC-202 (with competent cells)
Kit contents:
pBP-AT Vector (5-10 mg/ml) 20 ul
6X Reaction Solution 50 ul
(1.2 M NaCl, 0.06 M MgCl2)
DH5 alpha Chemically Competent Cells 20 x 50 ul
Storage:
pBP-AT Vector and X-gal -20oC or lower
DH105 alpha Chemically Competent Cells -80oC
6X Reaction Solution 4oC or lower
Protocol:
1. Thaw a vial of chemically competent cells on ice, and place S.O.C. medium at room temperature.
2. Warm up a 10 cm LB plus 100 ug/ml kanamycin agar plate to room temperature. Spread 30 ml of 40 mg/ml X-gal on the plate. Keep the plate at 37oC.
3. Set up the ligation reaction by adding the reagents in the following order:
PCR product 0.5-4 ul (containing 5-50 ng DNA)
6X Reaction Solution 1 ul
Sterile water add to a final volume of 5 ul
pBP-AT Vector 1 ul
Total volume 6 ul
4. Mix the reaction gently and incubate at room temperature for 5 minutes. Then place the ligation reaction on ice.
5. Add 2 ul of the ligation reaction to the pre-thawed competent cells and mix gently. Avoid pipetting up and down.
6. Incubate on ice for 30 minutes.
7. Heat-shock the cells at 42oC for 30 seconds. Immediately place the cells on ice for 1-2 minutes.
8. Add 250 ul of room temperature S.O.C. to the cells. Cap the vial and shake it at 200 rpm, 37oC for 1 hour.
9. Spread 50-250 ul of the transformation on the pre-prepared LB agar plate.
10. Pick white colonies and grow them individually in a proper medium.
Note:
1. pBP-AT Vector can be used to clone PCR product from tens of bases to at least 5 kb .
2. PCR product must be generated with Taq DNA polymerase. So the two 3’ ends of the PCR product have an unpaired base A.
3. PCR primers for PCR reaction must not have 5’ phosphate.
4. pBP-AT Vector contains the lacZ-a fragment gene. In the presence of X-gal, the transformants are blue. When PCR product is inserted into the vector, it will disrupt the expression of lacZ-a fragment and the transformants are white.
5. For best results, PCR product less than 2 kb should be column-purified to remove primers and primer-dimers. If longer than 2 kb, it should be gel-purified. Adding PCR product directly to the ligation reaction without purification will increase the background colony number, and thus blue/white screening is essential.
6. If PCR product is copied from a plasmid which contains a kanamycin resistant gene, treat the PCR product with Dpn I before purification to destroy the template.
7. For best results, PCR product should be freshly prepared.
Technical information:
The flanking sequences of the cloning sites:
Kpn I Sac I BamH I BamH I EcoR V
1 caagcttggt accgagctcg gatccactag taacggccgc cagtgtgctg gaattgCccc Tt PCR a agggGcaatt cGGATCCgat atccatcaca
GTTcgAACCA TGGCTCGAGC CTAGGTGATC ATTGCCGGCG GTCACACGAC CTTAACGGGG AA PRODUCT T TCCCcGTTAA GCCTAGGCTA TAGGTAGTGT
Not I T7 Promoter
94 CTggcggccgct cgagcatgca GCTCGTACGT tctagagggc ccaattcgcc ctatagtgag tcgtattac
GACCGCCGGCGA GCTCGTACGT CGAGCATGCA AGATCTCCCG GGTTAAGCGG GATATCACTC AGCATAATG
Topoisomerase site 1 62
Topoisomerase site 2 63
T7 promoter (c) 137-153
T7 primer (c) 134-153
M13 (-20) forward primer (c) 161-176
M13 (-40 forward primer (c) 180-196
f1 origin 317-755
Kan promoter 951-1088
Kanamycin resistant gene 1089-1883
pUC origin 2562-3235
lac promoter 3452-3573
lac repressor binding site 3536-3556
M13 reverse primer 3562-3578
(c) = complementary strand
Trouble shooting:
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Problem
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Possible cause
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Solution
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Few or no colonies, but a positive control plasmid yielded normal number of colonies
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Did not use Taq DNA polymerase in PCR reaction
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Perform PCR with Taq DNA polymerase.
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Although used Taq DNA polymerase for PCR but not enough PCR product has 3' A overhangs.
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Taq DNA polymerase adds an A most efficiently next to a C. So you may need to redesign PCR primers and let the 5' end be a G.
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PCR primers have 5' phosphate.
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Do not use 5' phosphorylated primers in PCR.
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Few or no colonies and even a positive control plasmid did not yield a normal number of colonies
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Competent cells have low transformation efficiency.
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Us fresh competent cells with a transformation efficiency > 108/mg pUC19 to perform transformation.
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Few white colonies and most of the colonies had a blue spot in the middle
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Too much primers or primer-dimers present in the ligation reaction.
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Purify PCR product to remove primers and primer-dimers.
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Few colonies containing the correct PCR product
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Too many nonspecific PCR products in the ligation reaction.
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Gel-purify the expected DNA band from PCR product.
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