Mouse Antibody Variable Region Gene Amplification Kit

Instruction manual

First Edition (Revised on April, 2016)

[PRODUCT INFORMATION]

This kit can be used for antibody variable region gene amplification in hybridoma, mouse peripheral blood mononuclear cells , mouse spleen cells and other tissues. In the early stage, extracted mRNA and oligo dt was used to reverse transcription into cDNA.. This kit improves component by expansionsystem, which guarantee the specificity of amplification and expand compatibility for all antibody genes. It can be used for amplification of mouse antibody gene in any hybridoma, amplification of all antibody genes in mice after immunization and construction of mouse antibody library.

[REAGENTS AND SPECIFICATION]

NO.	Name	Standard
IS088-1	Mouse antibody heavy chain amplification system	100μL
IS088-2	Mouse antibody light chain amplification system	100μL
IS088-3	Taq DNA polymerase premixing system	500μL
IS088-4	Positive control	5μL

[APPLICATION]

Mouse antibody variable region gene amplification.

[STORAGE AND DEADLINE]

Store at -20 degrees, and shelf life is 6 months.

[EXPERIMENTAL STEPS]

After all reagents melting, centrifuge them instantly and place on ice. Prepare reaction system according to following components. (Take 10ul as an example)

1. Heavy chain amplification

NO.	Volume(ul)
MU-VH-MIX（VIAL 1#）	1
DNA POLYMERASE MIX	5
cDNA	a（contain 2-100ng DNA，about 0.2-1μL）
H2O	4-a

NOTICE：If you need positive control, change cDNA with 1ul positive control (IS088-4).

2. Light chain amplification

NO.	Volume(ul)
MU-VH-MIX（VIAL 2#）	1
DNA POLYMERASE MIX	5
cDNA	a（contain 2-100ng DNA，about 0.2-1μL）
H2O	4-a

Tip：If you need positive control, change cDNA with 1u positive control (is088-4).

 

3. Amplification according to the following conditions

Temp	Time	Cycles
94℃	3min	1
94℃	30s	30
55℃	30s
72℃	45s
72℃	5min	1
4℃	∞	1

4. The PCR products are used for 2% agarose electrophoresis. Take DNA band about 350bp as a template to expand and recycle by Sub-PCR. Antibody variable region gene are obtained after TA cloning and sequencing.

[IMPORTANT NOTES]

1. Avoid repeated freezing / thawing cycle, otherwise it will influence amplification efficiency.;

2. Prefer cDNA of fresh sample as template;

3. The preparation of reaction system should be on the ice.