InMap Chromosome step kit

This product is a kit for obtaining the unknown sequences of the flanks of genomic DNA based on the known sequences on the genomic DNA, which is improved on the principle of TAIL-PCR (Thermal Asymmetric Interlaced PCR). This method is characterized by high efficiency, high specificity, high sensitivity and easy operation compared with other traditional chromosome stepping methods (e.g. reverse PCR, junction method, etc.).
The flanking sequences of the known sequences were obtained after three rounds of PCR amplification using the four concatenated primers provided in the kit, i.e., TP Primer 1 (TP1), TP Primer 2 (TP2), TP Primer 3 (TP3), and TP Primer 4 (TP4), respectively, and paired with the specific primers designed according to the known sequences, i.e., Specific Primer 1 (SP1), Specific Primer 2 (SP2), and Specific Primer 3 (SP3) for thermal asymmetric PCR amplification. Specific Primer 1 (SP1), Specific Primer 2 (SP2) and Specific Primer 3 (SP3) were used for thermal asymmetric PCR amplification, and the flanking sequences of the known sequences could be obtained after three rounds of PCR amplification, and the flanking sequence could be obtained according to the sequence information obtained in the first step if the length obtained in a single experiment could not satisfy the experimental requirements.
This product is paired with a superior performance L-Exp Taq HS DNA Polymerase, with the ability to amplify long fragments or complex fragments with high GC content, is paired with a carefully optimized Buffer system to make the reaction highly specific, while at the same time obtaining longer target sequences with a guaranteed amplification success rate.
Control Template and Control Specific Primer are provided to facilitate control experiments.

individual parts making up a compound	norm
L-Exp Taq HS DNA Polymerase (5 U/µl)	25 μl
5X L-Exp Taq PCR Buffer (Mg)2+ plus)	500 μl
dNTP Mix (10 mM each)	100 μl
TP Primer 1 (100 µM)	30 μl
TP Primer 2 (100 µM)	30 μl
TP Primer 3 (100 µM)	30 μl
TP Primer 4 (100 µM)	60 μl
Control Template (200 ng / µl)	10 μl
Control Specific Primer 1 (10 µM)	10 μl
Control Specific Primer 2 (10 µM)	10 μl
Control Specific Primer 3 (10 µM)	10 μl

 

Storage temperature: -20℃ storage
Transportation temperature: dry ice transportation or -20℃ ice bag transportation

The Control Template was used as a template, and TP Primer 4 in this product was used to obtain the mouse genome. ApoE The 5′ sequence of the gene, electrophoresis results showed that after three rounds of PCR, a relatively single target band was amplified.

(1) First round of PCR

① Prepare the reaction solution:

② Reaction conditions:
Refer to 1 in the “Methods” for the first round of PCR reaction program, in which the annealing temperature is selected to be 65℃ and the extension time is selected to be 2 min.

(2) Second round of PCR

① Prepare the reaction solution:

② Reaction conditions:
Refer to 2 in the “Methods” for the second PCR reaction program, in which the annealing temperature is selected to be 65°C and the extension time is selected to be 2 min.

The electrophoresis results are shown below:

(4) Product analysis and recovery

utilization SteadyPure The DNA gel recovery kit (Code No. AG21005) was used to cut and recover the third round of PCR amplification products, followed by DNA sequencing using Control Specific Primer 3;
Sequencing results showed that the 5 kb DNA fragments obtained contained the ApoE Sequence of the 5’ end of the gene.