TaKaRa
Á¦Ç°¸í/Á¦Ç°ÄÚµå
Å°¿öµå°Ë»ö
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miRNA µî ÀúºÐÀÚ RNAÀÇ Cloning¿¡ ÃÖÀû

small RNA Cloning Kit

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
RR065
small RNA Cloning Kit
°ü·ÃÇмú ±¸¸ÅÇϱâ
10 ȸ
(Package)
914,000¿ø  RR065_e.v2008Da.pdf

  • ÀúºÐÀÚRNA·ÎºÎÅÍ °£ÆíÇÏ°í È¿À²ÀûÀ¸·ÎcDNA ÇÕ¼º
  • ÁõÆøÇÑ cDNA´Â °ð¹Ù·Î TA cloning °¡´É
Á¦Ç° ¼³¸í
¼¼Æ÷¿¡¼­ ¹ßÇöÇÏ´Â RNA Áß ´Ü¹éÁúÀ» ÄÚµåÇÏÁö ¾Ê´Â °ÍÀÌ ¸¹ÀÌ Á¸ÀçÇÏ°í ±×µéÀÌ Áß¿äÇÑ ±â´ÉÀ» °¡Áø °ÍÀ¸·Î ¹àÇôÁ³´Ù. À̵éÀº ±â´É¼º RNA¶ó´Â ¸íĪÀ¸·Î ºÐ·ùµÇ¾î ÀÖ°í ±× Áß¿¡¼­µµ ƯÈ÷ 18~26 ¿°±â Á¤µµÀÇ ÀúºÐÀÚ RNA (small RNA, miRNA)ÀÇ ±â´ÉÀÌ ÁÖ¸ñ ¹Þ°í ÀÖ´Ù. ÀÌ ÀúºÐÀÚ RNAÀÇ ±â´ÉÀº ¾Æ·¡¿Í °°´Ù.
(1) À¯ÀüÀÚ ¹ßÇöÀ» mRNA ¼öÁØ¿¡¼­ ¾ïÁ¦
(2) À¯ÀüÀÚ ¹ßÇöÀ» translation ¼öÁØ¿¡¼­ ¾ïÁ¦

ÀúºÐÀÚ RNAÀÇ ¹ßÇö Çؼ®ÇÏ´Â ¼ö´ÜÀÇ Çϳª·Î Ŭ·Î´×¿¡ ÀÇÇÑ Çؼ®ÀÌ ÀÖ´Ù. ÀúºÐÀÚ RNA´Â mRNA¿Í°°Àº polyA tailÀÌ ¾ø±â ¶§¹®¿¡ ºÐ¸®µÈ ÀúºÐÀÚ RNAÀÇ 5'°ú 3'¸»´Ü¿¡ RNA ¶Ç´Â RNA/DNA chimera ¾î´ðÅ͸¦ °áÇÕ½ÃÄÑ ¿ªÀü»ç ¹ÝÀÀ ÈÄ, PCR·Î ÁõÆøÇÏ¿© Ŭ·Î´×ÇÏ´Â ¹æ¹ýÀÌ ÀÌ¿ëµÇ°í ÀÖ´Ù. º» Å°Æ®´Â À§ÀÇ ¿ø¸®¸¦ ¹ÙÅÁÀ¸·Î ÇÑ ÀúºÐÀÚ RNAÀÇ Å¬·Î´×¿ë cDNA ÁõÆø Å°Æ®ÀÌ´Ù. ÀÌ Å°Æ®´Â ¸¶±×³×ƽ bead¸¦ ÀÌ¿ëÇÏ¿© º¹ÀâÇÑ ÀÛ¾÷À» ÃÖ´ëÇÑ Á¦¿ÜÇÏ°í °£ÆíÇÑ Á¶ÀÛÀ¸·Î ÀúºÐÀÚ RNA À¯·¡ cDNA¸¦ ÁõÆøÇÒ ¼ö ÀÖµµ·Ï °í¾ÈµÇ¾ú´Ù. ÁõÆøµÈ cDNA´Â °ð¹Ù·Î TA Ŭ·Î´×¿¡ ÀÌ¿ëÇÏ´Â °ÍÀÌ °¡´ÉÇÏ´Ù. ¶ÇÇÑ ¾î´ðÅÍ¿¡ ÀÖ´Â Á¦ÇÑ È¿¼Ò »çÀÌÆ®¸¦ ÀÌ¿ëÇÏ¿© Ŭ·Î´×µµ °¡´ÉÇÏ´Ù.

1. Á¶Á¦ÇÑ small RNA¿¡ BAP ó¸®ÇÏ¿© 5'ÀÇ Àλê±â¸¦ Á¦°ÅÇÑ´Ù.
2. BAP ó¸®ÇÑ RNAÀÇ 3'¸»´Ü¿¡ biotinÈ­µÈ RNA/DNA chimera ¾î´ðÅ͸¦ °áÇÕ½ÃŲ´Ù.
3. Streptavidin ¸¶±×³×ƽ bead¿Í biotinÈ­µÈ ¾î´ðÅ͸¦ °áÇÕ½ÃÄÑ small RNA¸¦ ȸ¼öÇÏ¿© T4 polynucleotide kinase·Î 5 '¿¡ Àλê±â¸¦ Ãß°¡ÇÑ´Ù.
4. 5'¿¡ RNA/DNA chimera ¾î´ðÅ͸¦ °áÇÕ½ÃŲ ÈÄ, RT-primer¸¦ ÀÌ¿ëÇÏ¿© ¿ªÀü»ç È¿¼Ò(M-MLV4)·Î ¿ªÀü»ç ¹ÝÀÀÀ» ¼öÇàÇÑ´Ù.
5. cDNA¸¦ bead¿¡¼­ ȸ¼öÇÏ¿© PCRÀ» ½Ç½ÃÇÑ´Ù.
6. PCR´ÜÆí ¶Ç´Â Á¦ÇÑ È¿¼Ò ó¸® ´ÜÆíÀ» ȸ¼öÇÏ¿© Ŭ·Î´×ÇÑ´Ù.
Á¦Ç°±¸¼º
Package 1

1.

RNase Inhibitor

40 U/¥ìl

50 ¥ìl

2.

10¡¿BAP Buffer

 

200 ¥ìl

3.

Alkaline Phosphatase (BAP)

 

20 ¥ìl

4.

0.1% BSA

0.1%

60 ¥ìl

5.

3' adaptor *1, *2

 

10 ¥ìl

6.

T4 RNA Ligation Buffer

 

600 ¥ìl

7.

T4 RNA Ligase

40 U/¥ìl

20 ¥ìl

8.

5¡¿T4 PNK Buffer

 

100 ¥ìl

9.

T4 Polynucleotide Kinase

10 U/¥ìl

10 ¥ìl

10.

5' adaptor *1

 

10 ¥ìl

11.

5¡¿RT Buffer

 

40 ¥ìl

12.

dNTP Mixture

°¢2.5 mM

90 ¥ìl

13.

PCR-R & RT-Primer *1, *3

50 pmol/¥ìl

20 ¥ìl

14.

M-MLV RTase (RNaseH free)

200 U/¥ìl

10 ¥ìl

15.

2¡¿PCR Buffer

 

250 ¥ìl

16.

PCR Primer F *1

50 pmol/¥ìl

10 ¥ìl

17.

TaKaRa Ex Taq HS

5 U/¥ìl

10 ¥ìl

18.

Control RNA *4

5 pmol/¥ìl

10 ¥ìl


Package 2

19.

3 M Sodium Acetate (pH5.2)

200 ¥ìl

20.

Dr. GenTLE Precipitation Carrier *5

80 ¥ìl

21.

2¡¿B/W Buffer

2.7 ml

22.

MAGNOTEX-SA

100 ¥ìl

23.

RNase-free dH2O

10 ml


*1¡¡

3' adaptor,5' adaptor,PCR Primer F¹×PCR-R & RT-Primer¿¡´Â,Sse8387 IÀÇ »çÀÌÆ®°¡ Ãß°¡µÇ¾î ÀÖ´Ù. PCR»ê¹°°ú º¤Å͸¦Sse8387 I°úPst I ·Î Àý´ÜÇÏ¿© ¶óÀÌ°ÔÀÌ¼Ç ÇÒ ¼ö ÀÖ´Ù. º» Å°Æ®¿¡´Â Á¦ÇÑÈ¿¼Ò¿Í Ŭ·Î´×¿ë º¤ÅÍ µîÀº Æ÷ÇÔµÇÁö ¾Ê±â ¶§¹®¿¡ º°µµ·Î Áغñ ÇØ¾ß ÇÑ´Ù.

*2¡¡

3' adaptor´Â biotinÈ­ µÇ¾î ÀÖ´Ù.

*3¡¡

PCR¿ëreverse primer, ¿ªÀü»ç primer·Î »ç¿ëÇصµ µÈ´Ù.

*4

Control RNA´Â 5¡¯ ¸»´ÜÀÌ ÀλêÈ­µÈ 21 mer RNAÀÌ´Ù.
*5 GenTLE Precipitation Carrier (code 9094)¿Í °°Àº Á¦Ç°ÀÌ´Ù.
º¸Á¸
Package 1 : -20 ¡É
Package 2£º 4 ¡É
Å°Æ® ÀÌ¿Ü¿¡ ÇÊ¿äÇÑ ½Ã¾à ¹× ±â±¸·ù
[½Ã¾à]
  • Phenol:chloroform:isoamylalcohol (25£º24£º1, v/v/v)
  • chloroform:isoamylalcohol (24:1, v/v)
  • ¿¡Åº¿Ã
  • TE¹öÆÛ(10 mM Tris-HCl,1 mM EDTA,pH8.0)
  • 0.1 N NaOH
  • DNA size marker
  • 20 bp DNA Ladder(code 3409)
  • RNA size marker
    14-30 ssRNA Ladder Marker(code 3416)
  • 10% polyacrylamide gel (¹Ìº¯¼º)
  • 15% polyacrylamide gel (º¯¼º)
  • Á¦ÇÑ È¿¼ÒSse8387 I (Code 1183)
  • Cloning vector
  • Competent cell
  • ¶óÀÌ°ÔÀÌ¼Ç¿ë ½Ã¾à
    DNA Ligation Kit<Mighty Mix>(code 6023)
  • small RNAÃßÃ⠽þà

[±â±¸]
  • ¸¶ÀÌÅ©·Î ¿ø½É±â
  • Ç׿ ¼öÁ¶(´ÙÀ½ÀÇ °¢ ¿Âµµ ¼³Á¤À¸·Î »ç¿ë£»È÷Æ®ºí·Ï »ç¿ë °¡´É)
    15¡É,25¡É,37¡É,42¡É,70¡É
  • ¸¶ÀÌÅ©·Î ÇÇÆêÆ®
  • ¸¶ÀÌÅ©·Î ¿ø½É Æ©ºê
  • Thermal cycler
  • PCR¿ë0.2 mlÆ©ºê
  • ÇÊÅÍ ÇÇÆêÆ®ÆÁ
  • Agarose gel ¹× Àü±â¿µµ¿ ÀåÄ¡
  • polyacrylamide Àü±â¿µµ¿ ÀåÁö
  • ¸¶±×³×ƽ ½ºÅĵå

Keyword :

small RNA Cloning
>
Æ÷À¯µ¿¹° ¼¼Æ÷ ¹ßÇö vector
pBApo-CMV vector ½Ã¸®Áî
>
Æ÷À¯µ¿¹° ¼¼Æ÷ ¹ßÇö vector
pBApo-EF1¥á Vector ½Ã¸®Áî
>
miRNA µî ÀúºÐÀÚ RNAÀÇ Cloning¿¡ ÃÖÀû
small RNA Cloning Kit
>
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small RNA Gel Extraction Kit