Dinh 2011/NOTES/2011-9-2

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T4 Polynucleotide Kinase[edit]

  • NEB kit:
T4 PNK (10U/ul)
PNK buffer (10x) - *PNK buffer has no ATP. 
  • Up 300 pmol of 5' termini in a 50 ul reaction containing 1X buffer and 10 Units of T4 PNK. The enzyme has 100% activity in T4 DNA Ligase (Quick Ligase buffer).

Phosphorylating the splint:

APV6_Splint (100uM)      3ul         (300pmol)
T4 PNK (10U/ul)          1ul          (10U)
H2O                      2.5ul
QuickLigase Buffer (2x)  6.5ul          (1x)
============================================
                         13ul
No enzyme control:
APV6_Splint (100uM)      3ul         (300pmol)
T4 PNK (10U/ul)          0ul          (10U)
H2O                      3.5ul
QuickLigase Buffer (2x)  6.5ul          (1x)
============================================
                         13ul
  • Need 330,000x25,000 molecules = 8.25 x 10^9 molecules of probes
 1.25 pmoles of probes will give ~90x of this required amount.

Phosphorylating the probes:

98ng/ul lambda exo digested probes, 2.15uM           6ul    (12.9pmol)
T4 PNK (10U/ul)                                    0.5ul      (5U)
QuickLigase Buffer (2x)                            6.5ul      (1x)
======================================================================
                                                   13ul
No enzyme control:
98ng/ul lambda exo digested probes, 2.15uM         6.5ul    
T4 PNK (10U/ul)                                      0ul      (5U)
QuickLigase Buffer (2x)                            6.5ul      (1x)
======================================================================
                                                   13ul
  • Purify both with 1.8x AMPure beads (24 ul) and elute with 60ul EB
  • Quantify with QUBIT ssDNA kit
 APV6_Splint-   0.23ng/ul  0.024uM
 APV6_Splint+P  0.27ng/ul  0.028uM
 DMR330KE-     12.53ng/ul  
 DMR330KE+P     9.99ng/ul
  • APV6_Splint (31nt) is 9.5kDa

Test circularization #5[edit]

Amount of template:   (splint to template)     # moles              Concentration
                              2:1            1.25x10^-12         0.041 uM (1.89 ng/ul)
*Increase the amount of dNTP to add:
Use 15000 moles of dNTP per 1 moles of template = 1.875x10^-8 moles of dNTP
10mmoles/L * 1.9 x 10^-6 L
Amount of splint: 2.5x10^-12 moles 

Set up for DMR330K probes:

Reaction using APV6_Splint phosporylated:
Linear template              30 ul
Ampligase buffer (10x)        5 ul
APV6_Splint (0.027uM)       9.3 ul
dNTP (10mM)                 1.9 ul
Stoffel (10U/ul)            0.5 ul
Ampligase (5U/ul)             1 ul
H2O                         2.3 ul
===================================
                             50 ul
Reaction using APV6_Splint no PNK:
Linear template              30 ul
Ampligase buffer (10x)        5 ul
APV6_Splint (0.024uM)      10.9 ul
dNTP (10mM)                 1.9 ul
Stoffel (10U/ul)            0.5 ul
Ampligase (5U/ul)             1 ul
H2O                         0.7 ul
===================================
                             50 ul
Template dilution:
(1) ~Lambda exo digested probes and PNK treated = 9.99 ng/ul 
9.99 ng/ul * X ul = 1.89 * 130 ul = 
    1) Add 24.6 ul probes + 105.4 ul H2O 
(2)~Lambda exo digested probes and -PNK treated = 12.53 ng/ul 
12.53 ng/ul * X ul = 1.89 * 130 ul = 
    1) Add 19.6 ul probes (98ng/ul) + 110.4 ul H2O 
4 conditions:
 a) with unphosporylated splint
 b) with phosphorylated splint
 c) with stoffel only + phosphorylated splint
 d) with ligase only + untreated splint
SLN enzyme mix:             1x    4.5x
dNTP (10mM)                1.90 -> 8.55
Ampligase buffer (10x)        5 -> 22.5 
Stoffel (10U/ul)           0.50 -> 2.25
Ampligase (5U/ul)          1.00 -> 4.5
------------------------------------------
                            Add 8.4 per reaction (a, b)

SN enzyme mix:
SLN enzyme mix:             1x    2.5x
dNTP (10mM)                1.90 -> 4.75
Ampligase buffer (10x)        5 -> 12.5 
Stoffel (10U/ul)           0.50 -> 1.25
H2O                        1.00 -> 2.5
------------------------------------------
                            Add 8.4 per reaction (c)
LN enzyme mix:              1x    2.5x
dNTP (10mM)                1.90 -> 4.75
Ampligase buffer (10x)        5 -> 12.5 
H2O                        0.50 -> 1.25
Ampligase (5U/ul)          1.00 -> 2.5
------------------------------------------
                            Add 8.4 per reaction (d)
Prepare strip tubes:
1a 1b 1c 1d = add 30 ul of template 1
2a 2b 2c 2d = add 30 ul of template 2
-Add 10.9 ul unphosphorylated splint (-PNK treatment) to a's + 0.7 ul H2O
-Add  9.3 ul phosphorylated splint (+PNK treatment) to b,c + 2.3 ul H2O
-Add 11.6 ul splint mix (2.5 ul 10uM untreated splint + 113.5 H2O) to d's 
Program:
94 C 3min -> (94C* 30s -> 58C 10 min)x5 -> 94C for 3 min to inactivate enzymes
Run TBU gel:
5ul of reaction each + 5 ul TBE-Urea 2x buffer = loaded 8ul
1 ul of low mass ladder + 5 ul H2O + 5 ul TBE-UREA 2x buffer = loaded 6ul and 3 ul 
File:ZhangLab 2 2011-09-06 19hr 35min.jpg
1a = template (PNK+) + splint (PNK-) + SLN
1b = template (PNK+) + splint (PNK+) + SLN
1c = template (PNK+) + splint (PNK+) + SN
1d = template (PNK+) + splint (untreated) + LN
2a = template (PNK-) + splint (PNK-) + SLN
2b = template (PNK-) + splint (PNK+) + SLN
2c = template (PNK-) + splint (PNK+) + SN
2d = template (PNK-) + splint (untreated) + LN


Discussion[edit]

  • I've made a mistake in using AMPure beads to purify the splint (they are only 31 nt long) so the yield was very low.
  • Also, I miscalculated the amount of splint to use - thus almost no splint was added to the reactions 1a, 1b, 1c, 2a, 2b, 2c
  • I can barely see any circle or concatemers products from the LN reaction - is the AmpLigase bad? Or is the phosphorylation not working? The same splint was used to get circle product before.
  • I need to repeat the splint T4 PNK reaction and I need to perform ethanol precipitation to purify the splints.
  • Stoffel is working
  • Splint is binding
  • T4 PNK not working ? - repeat with new reagents
  • AmpLigase not working ? - repeat with new reagents from Noi