時間：2020-08-05 10:38:00 瀏覽：3693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:
? Water control: No DNA template added.
? 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. 配種與繁殖匯總
小鼠配種與繁殖情況如下圖所示。
（1）+/+為DNA雙鏈都含有XXX基因，即野生型小鼠；+/-為只有一條DNA鏈含有XXX基因，即雜合子小鼠；-/-為DNA雙鏈都不含XXX基因，即純合子小鼠。
（2）紅色字體標記表示純合子小鼠，藍色字體標記表示小鼠已死亡。
PCR鑒定
（1）5/9，2#，6#鑒定。
根據圖一，說明：2-1、6-1用引物F1/R1進行PCR；2-2、6-2用引物F1/R2進行PCR。鑒定結果：2#，6#為雜合子。
（2）5/19，7#~13#鑒定。
根據圖二，說明：前面是用引物F1/R1進行PCR；后面是用引物F1/R2進行PCR。鑒定結果：7#、9#、10#為純合子；11#、12#、13#為雜合子；8#為野生小鼠。其中11#引物F1/R1條帶不明顯，后續重新取材鑒定。
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