biopioneerinc/Blunt End PCR Cloning Kit without Competent Cells, 20 reactions/1/PCK-101,biopioneerinc,,biopioneerinc,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：biopioneerinc/Blunt End PCR Cloning Kit without Competent Cells, 20 reactions/1/PCK-101

产品编号：PCK-101

市场价：¥5200.00

场地：美国(厂家直采)

产品分类：分子类>分子诊断>定量PCR>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$260.00

品牌： biopioneerinc

公司：biopioneerinc

公司分类：

商品介绍

BP-Blunt ^TMCloning Kit

For cloning PCR product generated with high fidelityDNA polymerase

Cat No. PKC-101 (without competent cells)

Cat No. PKC-102 (with competent cells)

Kit contents:

pBP-Blunt Vector (5-10 mg/ml) 20 ul

6X Reaction Solution50 ul

(1.2 M NaCl, 0.06 M MgCl2)

DH5 alpha Chemically Competent Cells 20 x 50 ul

Storage

pBP-Blunt Vector and X-Gal -20^oC or lower

DH5alphaChemically Competent Cells -80^oC

6X Reaction Solution4^oC 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 40 ml of 40 mg/ml X-gal on the plate. Keep the plate at 37^oC.

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 Solution1 ul

Sterile wateradd to a final volume of 5 ul

pBP-Blunt 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 42^oC 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, 37^oC 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-Blunt Vector can be used to clone PCR product from tens of bases to at least 5 kb .

2.PCR product must be generated with high fidelity DNA polymerase, and thus the PCR product has blunt ends. For best results, do not use DNA polymerase reagent containing Taq DNA polymerase.

3.PCR primers for PCR reaction must not have 5’ phosphate.

4.pBP-Blunt Vector contains the lacZ-a fragment gene. In the presence of X-gal, the transformants are blue. When inserted into the vector, PCR product will disrupt the expression of lacZ-a fragment and the transformants are white.

5.For best results, PCR product shorter 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 containing a kanamycin resistant gene, treat the PCR product with Dpn I before purification to destroy the template.

7.For best cloning results, PCR product should be freshly prepared.

Technical information:

The flanking sequences of the cloning sites:

Kpn ISac I BamH I BamH I EcoR V

1caagcttggt accgagctcg gatccactag taacggccgc cagtgtgctg gaattgCccc Tt PCRaagggGcaatt cGGATCCgat atccatcaca

GTTcgAACCA TGGCTCGAGC CTAGGTGATC ATTGCCGGCG GTCACACGAC CTTAACGGGG AA PRODUCTT TCCCcGTTAA GCCTAGGCTA TAGGTAGTGT

Not I T7 Promoter

94 CTggcggccgct cgagcatgca GCTCGTACGTtctagagggc ccaattcgcc ctatagtgag tcgtattac

GACCGCCGGCGA GCTCGTACGT CGAGCATGCA AGATCTCCCG GGTTAAGCGG GATATCACTC AGCATAATG

Topoisomerase site 162

Topoisomerase site 263

T7 promoter (c)137-153

T7 primer134-153

M13 (-20) forward primer161-176

M13 (-40 forward primer 180-196

f1 origin 317-755

Kan promoter 951-1088

Kanamycin resistant gene 1089-1883

pUC origin2562-3235

lac promoter 3452-3573

lac repressor binding site3536-3556

M13 reverse primer3562-3578

Trouble shooting:

Problem	Possible cause	Solution
Few or no colonies, but a positive control plasmid yielded normal number of colonies	Did not use high fidelity DNA polymerase in PCR.	Perform PCR with high fidelity DNA polymerase.
	PCR primers have 5" phosphate.	Do not use 5" phosphorylated primers in PCR.
Few or no colonies and even a positive control plasmid did not yield a normal number of colonies	Competent cells have low transformation efficiency.	Us fresh competent cells with a transformation efficiency > 10⁸/mg pUC19 for transformation.
Few white colonies and most of the colonies had a blue spot in the middle	Too much primers or primer-dimers present in the ligation reaction.	Purify PCR product to remove primers and primer-dimers.
Few colonies containing the correct PCR product	Too many nonspecific PCR products in the ligation reaction.	Gel-purify the expected DNA band from PCR product.