时间：2020-08-05 10:38:00 浏览：6634次
1. Product Information
Name
C57BL/6-XXXtm1cyagen
Serial Number
KOCMP-21018-XXX
Gene
XXX
NCBI ID
18024
Strain
C57BL/6
Type
conventional knockout
2. gRNA target sequence
gRNA1 (matching forward strand ofgene): CAGCCCTTGGTTAGCAGCGCAGG
gRNA2 (matching forward strand of gene): TTCTATACTAAACGTTAGCTAGG
3. Delivery Information
4. Genotyping Strategy
5. PCR Screening
Animals 2 and 6 were identified positive by PCR screening.
Note:
1)PCR was carried out in 25 µL volume for 35 cycles under standard conditions, with primers listed above added to each reaction.
2) Taq DNA polymerase used was P112-01.
3) Two controls used in PCR genotyping are:
Ø Water control: No DNA template added.
Ø Wildtype control: 400 ng of mouse genomic DNA.
6. Sequencing Confirmation
Sequencing Primer for PCR product:
Sequence primer (F1): 5’-GAAGCATTATCATTTGGTGGGAAC-3’
Sequencing Results:
Positive animals
Mouse ID: 2, 6 (Deleted 4961 bp and inserted 28 bp)
7. Breeding and Genotyping strategy
8. 配种与繁殖汇总
小鼠配种与繁殖情况如下图所示。
（1）+/+为DNA双链都含有XXX基因，即野生型小鼠；+/-为只有一条DNA链含有XXX基因，即杂合子小鼠；-/-为DNA双链都不含XXX基因，即纯合子小鼠。
（2）红色字体标记表示纯合子小鼠，蓝色字体标记表示小鼠已死亡。
PCR鉴定
（1）5/9，2#，6#鉴定。
根据图一，说明：2-1、6-1用引物F1/R1进行PCR；2-2、6-2用引物F1/R2进行PCR。鉴定结果：2#，6#为杂合子。
（2）5/19，7#~13#鉴定。
根据图二，说明：前面是用引物F1/R1进行PCR；后面是用引物F1/R2进行PCR。鉴定结果：7#、9#、10#为纯合子；11#、12#、13#为杂合子；8#为野生小鼠。其中11#引物F1/R1条带不明显，后续重新取材鉴定。
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