XL10-Gold感受态细胞

Improved Transformation of Large DNA

Stratagene’s XL10-Gold® ultracompentent cells^* were created to transform large DNA molecules with high efficiency. These cells exhibit the Hte phenotype,^** which increases the transformation efficiency of large and ligated DNA molecules. In addition, the XL10-Gold strain allows blue-white color screening, cloning of methylated DNA and produces high-quality miniprep DNA. XL10-Gold ultracompetent cells are the host cells of choice for cloning experiments that require high transformation efficiencies.

Transformation of Ligated DNA

The LIC method was used to anneal the pCAL-n-FLAG vector (28 ng) to the kanamycin-resistance gene in a 15-µl reaction volume. Of this reaction, 1-µl aliquots were used to transform 100 µl of competent cells according to the manufacturers’ protocol. The entire transformation was spread onto LB agar plates with kanamycin.

Plasmid Libraries Approach Lambda Quality

The XL10-Gold ultracompetent cells are ideal for constructing plasmid libraries. Ligated plasmid DNA generally transforms with significantly lower efficiency than supercoiled plasmids, and large plasmids transform less efficiently than small plasmids.  This bias against transformation of larger constructs impacts the construction of plasmid libraries and reduces the probability of finding full-length cDNA clones. XL10-Gold ultracompentent cells decrease this size bias and produce larger and more complex libraries, approaching the quality and efficiency of lambda libraries. Stratagene offers three plasmid cDNA library construction kits for directional cloning, supplied with XL10-Gold ultracompetent cells for superior performance.

Restriction Minus for Cloning Methylated DNA

Stratagene’s XL10-Gold cells make it possible to clone methylated DNA.  Eukaryotic genomic DNA can be highly methylated, and the methylated patterns can vary in different tissues and at different times during development. cDNA is often methylated during synthesis to protect internal restriction sites from cleavage during later processing.  When DNA is methylated in a fashion unlike the bacterial host patterns, it is cleaved by the E. coli host restriction systems, resulting in a significantly skewed representation.

The XL10-Gold cells are deficient in all known E. coli K12 restriction systems to decrease the likelihood of generating unrepresentative libraries. Absence of these endogenous bacterial restriction systems increases the efficiency of introducing eukaryotic DNA into E. coli, and increases the size and representation of libraries constructed with methylated or hemi-methylated DNA.
菌种培养及打管说明：
1、安瓿瓶开封：用浸过75％酒精的脱脂棉擦净安瓿管，用火焰加热其顶端，滴少量无菌水至加热顶端使之破裂，用锉刀或者镊子敲下已破裂的安瓿管顶端。
2、菌株恢复培养：用无菌吸管吸取0.3--0.5ml适宜的液体培养基，滴入安瓿管内，轻轻振荡，使冻干菌体溶解呈悬浮状。吸取全部菌体悬浮液，移植于1-2支建议的培养基试管中，并在建议的条件下培养。
3、注意事项：菌种活化前，请将安瓿管保存在6-10℃的环境下，某些菌种经过冷冻干燥保存后，延迟期较长，需要连续两次继代培养才能正常生长
4、复苏后的菌种在传1-2代后使用。
5、暂不启开的安瓿及复苏后需保藏的斜面应于4℃中保藏。
特别注意事项：
l、微生物菌种应保藏于低温、清洁和干燥的地方,室温放置时问过长会导致菌种衰退;
2、菌种操作应在无菌条件下进行,防止杂菌污染:
3、斜面菌种保藏时间通常为1-2个月,应根据菌种状况及时转接;冻干菌种保藏时间通常为5~10年;
4、菌种使用过程中如出现杂菌污染或菌种生产性能下降，应及时与我司销售联系或更换新的菌种。