PCR克隆篩選試劑盒
產(chǎn)品名稱：PCR克隆篩選試劑盒
英文名稱：PCR Quick Screening Kit
貨號：K903-500
品牌：原裝進口
包裝：500次分析
保存建議: 請在接收到該Biovision品牌的PCR克隆篩選試劑盒產(chǎn)品后，置于-20℃保存.
產(chǎn)品特色：1、操作快速簡單；2、克隆可直接通過瓊脂糖凝膠電泳分析.
Assay Procedure:
1. Add 1 μl of InsertFinder Lysis Buffer to the bottom of each labeled PCR tube.
2. Pick a fresh single clone of transformants (bacteria or yeast) with a pipet tip (do not
use tooth picks). Carefully transfer a small amount of the colony into the 1 μl of Lysis
Buffer prepared in step 1. The buffer will become cloudy.
3. Prepare a reaction Master Mix. For each clone, mix the follows:
2 μl 10X PCR Buffer
2 μl dNTP
0.05 μg Primer 1
0.05 μg Primer 2
0.9 μl PCR Enhancer
dH2O to a total 19 μl
0.2 μl Taq Polymerase
Notes: a) We suggest including a positive control with a known insert and a negative
control without clone added.
b) Primer pairs can both be complementary to the insert, or to the cloning
vectors, or in combinations. If using the combination of primers from insert
and vector, insert orientation can be selected specifically.
4. Add 19 μl of the Master Mix into each PCR tube containing lysed clones.
5. Run PCR reaction 25 to 35 cycles* as follows:
94°C 30 sec.
55°C 120 sec.**
72°C 60 - 180 sec.***
Notes:
* We suggest running 25-30 cycles for high copy number plasmid and 30-35 cycles
for low copy numbers of plasmid.
**We suggest using an annealing temperature of 55°C. However, you may use a
suggested annealing temperature for your specific primer pairs.
***We recommend using a 60 sec elongation time for an insert < 1 kb, and 180 sec
for an insert > 1 kb.
6. Add DNA loading buffer into each sample and load 15 μl of the sample on a standard
agarose gel to determine which clones contain the proper DNA insert.
Note: For small inserts (< 300 bp), we suggest using Orange G DNA Loading Buffer
(BioVision Cat.# 2110-10) which offers better resolution of DNA fragments on
agarose gel.
如與英文說明書有異，以英文說明書為準。
PCR克隆篩選試劑盒說明書具體咨詢咨詢
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