基本描述

别名

HiFi II M-MLV (H-) 逆转录酶

规格或纯度

10000 U

英文名称

HiFi II M-MLV (H-) Reverse Transcriptase

储存温度

-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

HiFi II M-MLV(H-)是经过突变的M-MLV基因利用大肠杆菌工程菌进行重组与表达的逆转录酶，该酶能够催化以RNA或DNA：RNA杂交链为模板的互补DNA聚合反应。经过突变的HiFi II M-MLV(H-)逆转录酶RNase H活性缺失，减少了逆转录反应中RNA的降解，更容易获得全长的cDNA。HiFi II M-MLV(H-)逆转录酶能在55℃合成第一条链 cDNA，提供更高的特异性，稳定性强，可以合成最大12 kb的cDNA，cDNA产量高。适用于第一链cDNA的合成、RT-PCR、RT-qPCR以及全长cDNA文库的构建等。

H665664	Component	10 KU	Storage
H665664A	HiFi II M-MLV(H-) (200 U/μL)	50 μL	-20℃. Avoid freeze/thaw cycle.
H665664B	5×SuperRT Buffer	1 mL	-20℃. Avoid freeze/thaw cycle.

活性定义：

以Poly（A）为模板，oligo（dT）为引物，在37℃条件下，10分钟内催化掺入1 nmol的dTTP所需酶量定义为一个活性单位(U)。

质量控制：

200 U的本酶和1 µg的16 S，23 S rRNA在37℃下反应1小时，RNA的电泳谱带不发生变化。

注意事项：

1．在操作过程中应避免RNase污染，防止RNA降解或实验中的交叉污染，建议在专门的区域进行RNA操作，使用专门的仪器和耗材，操作人员带口罩和一次性手套并经常更换手套。

2．实验尽量使用一次性塑料器皿，若使用玻璃器皿，应使用0.1％DEPC（焦碳酸二乙酯）水溶液在37℃处理12小时，并在120℃下高压灭菌30分钟后使用，或者将玻璃器皿在180℃下干热灭菌60分钟后使用。实验中用到的无菌水应使用0.1%的DEPC 处理后进行高压灭菌。

3．本试剂盒中的所有试剂使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。所涉及的酶类使用后应尽快放回-20℃，避免反复冻融。

4．若起始RNA的量小于50 ng，建议加入RNA酶抑制剂（RNasin）。本试剂盒并未提供。

使用方法：

注意：10 ng-5 μg总RNA可建立20 μL反应体系，如果总RNA量大于5 μg，请按比例扩大反应体系。

i 逆转录操作步骤：

1．将RNA模板、引物、dNTP Mix、SuperRT Buffer、HiFi II M-MLV(H-)和RNase-Free Water溶解并置于冰上备用。

2．根据以下表格配制反应体系，总体积为20 μL。

试剂	20 μl反应体系	终浓度
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Oligo-dT Primer，100 µ M 或 Random Primers ，50 μ M 或 Specific Primer , 10 μ M	1 μl	/
RNA Template	X μl	1 ng-5 µg
5×SuperRT Buffer	4 μl	1 ×
HiFi II M-MLV(H-) (200U /μL)	0.5-1 μL	/
RNase-Free Water	up to 20 μL	/

注意：若起始RNA的量小于50ng，则建议加入RNA酶抑制剂（RNasin）。本试剂盒并未提供。

3．涡旋震荡混匀，短暂离心，使管壁上的溶液收集到管底。

4．55℃孵育1-30分钟，85℃孵育5分钟。反应结束后，短暂离心，置于冰上冷却。

5．逆转录产物可直接用于PCR反应和荧光定量PCR反应，或置于-20℃长期保存。

ii 若逆转录效率低，或RNA模板二级结构复杂、GC含量高时，建议采用以下步骤：

1．将RNA模板、引物、dNTP Mix、SuperRT Buffer、HiFi II M-MLV(H-)和RNase-Free Water溶解并置于冰上备用。

2．根据以下表格配制反应体系，总体积为15 μL 。

试剂	20 μl反应体系	终浓度
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Oligo-dT Primer，100 µ M 或 Random Primers ，50 μ M 或 Specific Primer , 10 μ M	1 μl	/
RNA Template	X μl	1 ng-5 µg
RNase-Free Water	up to 15 μL	/

3. 70℃孵育10分钟，迅速冰浴2分钟。

4. 短暂离心，使管壁上的溶液收集到管底。

5. 向以上反应液中加入4 μL 5×SuperRT Buffer。

注意：若起始RNA的量小于50 ng，则建议加入RNA酶抑制剂(RNasin)。本试剂盒并未提供。

6. 轻轻吹打混匀，若反转录引物为Oligo-dT Primer或Specific Primer时，

7. 42℃孵育2 分钟；若反转录引物为Random Primers，则25℃孵育10分钟。

8. 加入1 μL HiFi II M-MLV(H-) (200 U/μL)，轻轻吸打混匀。 55℃孵育50分钟。 85℃孵育5分钟。反应结束后，短暂离心，置于冰上冷却。

9. 逆转录产物可直接用于PCR反应和荧光定量PCR反应，或置于-20℃长期保存。

HiFi II M-MLV (H -) is a reverse transcriptase that recombines and expresses mutated M-MLV genes using E. coli engineering bacteria. This enzyme can catalyze complementary DNA polymerization reactions using RNA or DNA: RNA hybrid strands as templates. The mutated HiFi II M-MLV (H -) reverse transcriptase RNase H activity is missing, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi II M-MLV (H -) reverse transcriptase can synthesize the first strand of cDNA at 55 ℃, providing higher specificity, strong stability, and can synthesize up to 12 kb of cDNA with high cDNA yield. Suitable for the synthesis of first stranded cDNA, RT PCR, RT qPCR, and construction of full-length cDNA libraries.

H665664	Component	10 KU	Storage
H665664A	HiFi II M-MLV(H-) (200 U/μL)	50 μL	-20℃. Avoid freeze/thaw cycle.
H665664B	5×SuperRT Buffer	1 mL	-20℃. Avoid freeze/thaw cycle.

Activity definition:
Using Poly (A) as a template and oligo (dT) as a primer, the enzyme required to catalyze the addition of 1 nmol of dTTP within 10 minutes at 37 ℃ is defined as one active unit (U).
Quality control:
200 U of this enzyme reacted with 1 µ g of 16 S, 23 S rRNA at 37 ℃ for 1 hour, and the electrophoresis band of the RNA remained unchanged.
Notes:
1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.
2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.
3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.
If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.

Usage:
Attention: 10 ng-5 μ G Total RNA can establish 20 μ L reaction system, if the total RNA amount is greater than 5 μ g. Please expand the reaction system proportionally.
i Steps for reverse transcription:
1. Dissolve the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them on ice for later use.
2. Prepare a reaction system according to the following table, with a total volume of 20 μ L.

Reagent	20 μlReaction system	Final concentration
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Oligo-dT Primer，100 µ M Or Random Primers ，50 μ M or Specific Primer , 10 μ M	1 μl	/
RNA Template	X μl	1 ng-5 µg
5×SuperRT Buffer	4 μl	1 ×
HiFi II M-MLV(H-) (200U /μL)	0.5-1 μL	/
RNase-Free Water	up to 20 μL	/

Note: If the initial amount of RNA is less than 50ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.
3. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe.
4. Incubate at 55 ℃ for 1-30 minutes, and incubate at 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.
5. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.
ii If the reverse transcription efficiency is low, or the RNA template secondary structure is complex and the GC content is high, the following steps are recommended:
1. Dissolve the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them on ice for later use.
2. Prepare a reaction system according to the following table, with a total volume of 15 μ L.

Reagent	20 μlReaction system	Final concentration
dNTP Mix，2.5 mM Each	4 μl	500 μM Each
Oligo-dT Primer，100 µ M Or Random Primers ，50 μ M or Specific Primer , 10 μ M	1 μl	/
RNA Template	X μl	1 ng-5 µg
RNase-Free Water	up to 15 μL	/

3. Incubate at 70 ℃ for 10 minutes and quickly ice bath for 2 minutes.
4. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.
5. Add 4 to the above reaction solution μ L 5 x SuperRT Buffer.
Note: If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.
6. Gently blow and mix well. If the reverse transcription primer is Oligo dT Primer or Specific Primer,
7. Incubate at 42 ℃ for 2 minutes; If the reverse transcription primer is Random Primers, incubate at 25 ℃ for 10 minutes.
8. Join 1 μ L HiFi II M-MLV (H -) (200 U/ μ L) Gently pat and mix well. Incubate at 55 ℃ for 50 minutes. Incubate at 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.
9. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

h665664_hifi ii m-mlv (h-) reverse transcriptase.pdf

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