Jie:LabNotes/ASE/2009-6-10: Difference between revisions

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==Making ds-cDNA for Hues6/Hybrids/BJ==
==Making ds-cDNA for Hybrids/BJ==


     * Total RNA Samples:
     * Total RNA Samples:
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Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit
Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit


                             A     B   c    
                             A       B     C    
   RNA                    6ul   3ul 8ul
   RNA                    6ul     2.5ul 10ul
   10X DNase I buffer      5ul   5ul 5ul
   10X DNase I buffer      5ul       5ul   5ul
   RNase-Free DNase I      2ul    2ul 2ul
   RNase-Free DNase I      2.5ul  2.5ul 2.5ul
   H2O                     37ul  40ul 35ul
   H2O                   36.5ul    40ul 32.5ul
   37C 10min  
   37C 10min  


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Reverse transcription
Reverse transcription


H was not DNase treated.
                   A      B      C       
 
  RNA            8ul    8ul    8ul     
                   A      B      C       D     E    F    G    H
  10mM dNTP      1ul    1ul    1ul     
  RNA            8ul    8ul    8ul    8ul    8ul  8ul  8ul  8ul
  50uM dT12-18    1ul    1ul    1ul     
  10mM dNTP      1ul    1ul    1ul    1ul    1ul  1ul  1ul  1ul
  50uM dT12-18    1ul    1ul    1ul    1ul    1ul  1ul  1ul  1ul
                
                
  65C 5min -> chill on ice for 1min
  65C 5min -> chill on ice for 1min
  Add   
  Add   
  10X RT buffer  2ul    2ul      2ul    2ul  2ul    2ul      2ul    2ul
  10X RT buffer  2ul    2ul      2ul     
  25mM MgCl2      4ul    4ul      4ul    4ul  4ul    4ul      4ul    4ul
  25mM MgCl2      4ul    4ul      4ul     
  0.1M DTT        2ul    2ul      2ul    2ul  2ul    2ul      2ul    2ul
  0.1M DTT        2ul    2ul      2ul     
  RNaseOUT        1ul    1ul      1ul    1ul  1ul    1ul      1ul    1ul
  RNaseOUT        1ul    1ul      1ul     
  Superscript III 1ul    1ul      1ul     1ul  1ul    1ul      1ul    1ul
  Superscript III 1ul    1ul      1ul  
  Incubate all tube at 50C for 50min.
  Incubate all tube at 50C for 50min.


[edit] 2nd strand synthesis
2nd strand synthesis


Cleanup the first-strand reactions with G-25 columns.
Cleanup the first-strand reactions with qiaquick columns.


  To the ~40ul reactions, add     
  To the 30ul reactions, add     
  10ul NEBuffer 2 10ul
  10ul NEBuffer 2   5ul
  10mM dNTP        5ul
  10mM dNTP        2.5ul
  DNA Pol I(10U/ul) 2ul
  DNA Pol I(10U/ul) 1ul
  RNaseH            2ul
  RNaseH            1ul
  H2O              adjust to 100ul
  H2O              10.5ul
  Incubate at 16C for 2h, purified with Qiaquick columns.
  Incubate at 16C for 2h, purified with Qiaquick columns.
  Yield (use RNA's absorbance):
 
  A: Hues6-ES: 50ng/ul x 30ul
  Yield (use DNA's absorbance):                     RNA
  B: Hues6-EB: 55ng/ul x 30ul
  A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul         18.1ng/ul
  C: Hybrid1-ES: 30ng/ul x 30ul
  B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul         63.4ng/ul
  D: Hybrid1-EB: 63ng/ul x 30ul
  C: BJ(520ng/ul):         94.4ng/ul x 30ul         72.3ng/ul
E: Hybrid2-ES: 58ng/ul x 30ul
 
F: Hybrid2-EB: 53ng/ul x 30ul
==capture with CES27k9bp==
G: BJ:         44ng/ul x 30ul
 
H: Hybrid1-ES: 56ng/ul x 30ul
                B    C   
  Sample      8ul  8ul 
  10x buffer  2ul  2ul   
  CES27k9bp    5ul  5ul 
  H2O          5ul  5ul
   
SLN mixture:  
 
                                        ddH2O  Ligase Buffer  Ligase  Amplitaq    dNTP 
  2U/ul Stoffel;0.5U/ulLigase;0.5uM dNTP  0.1ul  0.1ul      0.1ul      0.2ul    (1uM)0.5ul   
                                x20      2ul      2ul        2ul        4ul    10ul
 
  95C 2min -> 60C 24h -> add 2ul SLN mix (dNTP 0.5uM, Stoffel 2U/ul, AmpLigase 1U/ul)
    -> 60C 18h -> 95C 5min -> 37c 1min -> add 2ul Exo I&III
    -> 37C 1h -> 94C 5min -> 4C hold
 
==PCR==
 
  Template                  10ul      x4
  2X iProof Mastermix    50ul      200ul
  AmpF6.2SoL (10uM)        4ul      16ul
  AmpR6.2SoL (10uM)        4ul      16ul 
  50X SYBG I            0.4ul      1.6ul
  H2O                  31.6ul    126.4ul
 
I did 20 cycles for BJ and 21 cycles for Hybrid_1.
 
[[Image:20090615_CES27k9bp_Hybrid1_BJ.jpg]]20090615_CES27k9bp_Hybrid1_BJ
 
Qiaquick purification and gel quantification:
 
Hybrid_1: 8.6ng/ul x 30ul
BJ: 14.3ng/ul x 30ul
 
I send the two samples to Zhen Ye as follows:
  Hybrid_1: 4.3ng/ul (37nM) x 5ul
  BJ: 7.2ng/ul (62nM) x 5ul

Latest revision as of 17:16, 18 June 2009

Making ds-cDNA for Hybrids/BJ[edit]

   * Total RNA Samples:
         o A: Hybrid1-ES: 750ng/ul
         o B: Hybrid1-ES: 1.9ug/ul
         o C: BJ: 520ng/ul
        

Clean up and concentrate all RNAs with Zymo DNA-Free RNA kit

                           A        B     C    
  RNA                     6ul     2.5ul  10ul
  10X DNase I buffer      5ul       5ul   5ul
  RNase-Free DNase I      2.5ul   2.5ul  2.5ul
  H2O                   36.5ul     40ul  32.5ul
  37C 10min 
   * Add 4 volumes of RNA binding buffer;
   * Transfer to Zymo-spin columns in collection tubes, spin @15k rpm for 30s;
   * Add 200ul RNA Wash buffer, spin @15k rpm for 30s;
   * Add 10ul DNase/RNase-free water, spin @15k rpm for 30s;

Reverse transcription

                 A       B       C      
RNA             8ul     8ul     8ul    
10mM dNTP       1ul     1ul     1ul     
50uM dT12-18    1ul     1ul     1ul     
              
65C 5min -> chill on ice for 1min
Add  
10X RT buffer   2ul     2ul      2ul     
25mM MgCl2      4ul     4ul      4ul     
0.1M DTT        2ul     2ul      2ul     
RNaseOUT        1ul     1ul      1ul     
Superscript III 1ul     1ul      1ul    
Incubate all tube at 50C for 50min.

2nd strand synthesis

Cleanup the first-strand reactions with qiaquick columns.

To the 30ul reactions, add    
10ul NEBuffer 2   5ul
10mM dNTP         2.5ul
DNA Pol I(10U/ul) 1ul
RNaseH            1ul
H2O              10.5ul
Incubate at 16C for 2h, purified with Qiaquick columns.
Yield (use DNA's absorbance):                     RNA
A: Hybrid1-ES(750ng/ul): 22.9ng/ul x 30ul         18.1ng/ul
B: Hybrid1-ES(1.9ug/ul): 80.2ng/ul x 30ul         63.4ng/ul
C: BJ(520ng/ul):         94.4ng/ul x 30ul         72.3ng/ul

capture with CES27k9bp[edit]

                B    C    
 Sample       8ul  8ul   
 10x buffer   2ul  2ul    
 CES27k9bp    5ul  5ul  
 H2O          5ul  5ul 

SLN mixture:

                                       ddH2O  Ligase Buffer   Ligase   Amplitaq    dNTP  
 2U/ul Stoffel;0.5U/ulLigase;0.5uM dNTP  0.1ul  0.1ul       0.1ul       0.2ul    (1uM)0.5ul    
                                x20      2ul      2ul         2ul         4ul     10ul
  95C 2min -> 60C 24h -> add 2ul SLN mix (dNTP 0.5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
    -> 60C 18h -> 95C 5min -> 37c 1min -> add 2ul Exo I&III
    -> 37C 1h -> 94C 5min -> 4C hold

PCR[edit]

 Template                  10ul       x4
 2X iProof Mastermix     50ul      200ul
 AmpF6.2SoL (10uM)        4ul       16ul
 AmpR6.2SoL (10uM)        4ul       16ul   
 50X SYBG I             0.4ul      1.6ul
 H2O                   31.6ul    126.4ul

I did 20 cycles for BJ and 21 cycles for Hybrid_1.

File:20090615 CES27k9bp Hybrid1 BJ.jpg20090615_CES27k9bp_Hybrid1_BJ

Qiaquick purification and gel quantification:

Hybrid_1: 8.6ng/ul x 30ul BJ: 14.3ng/ul x 30ul

I send the two samples to Zhen Ye as follows:

 Hybrid_1: 4.3ng/ul (37nM) x 5ul
 BJ: 7.2ng/ul (62nM) x 5ul