Dinh 2011/NOTES/2011-9-21

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DMR330K E-mix with 5/Phos/ primers[edit]

  • Noi amplified 1 plate of DMR330K-E mix with 5' phosphorylated (Forward primer with 3 phosphothrioate bonds / Reverse primer with no phosphothrioate bonds) primers
  • Ethanol purification purified with 12 tubes
  • Column purification with 12 columns, Nanodrop: 148.8 ng/ul x 530ul = 78.8 ug
  • Lambda exo: 16ul 10x buffer, 134ul of probes, 10ul of Lambda exo
  • Column purification with 4 columns, Nanodrop: 106.2ng/ul x 160ul = 17.0 ug

T4 Polynucleotide Kinase repeat[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:

102.6ng/ul lambda exo digested probes, 2.33uM       11ul  (~25.63pmol)
T4 PNK (10U/ul)                                    1.0ul      (5U)
QuickLigase Buffer (2x)                             12ul      (1x)
======================================================================
                                                    24ul
No enzyme control:
102.6ng/ul lambda exo digested probes, 2.33uM       11ul    
T4 PNK (10U/ul)                                      0ul      (5U)
QuickLigase Buffer (2x)                             11ul      (1x)
======================================================================
                                                    22ul
  • Incubate at 37C for 40 minutes
  • Ethanol purification, elute with 50ul EB
  • Quantify with QUBIT ssDNA kit
 Splint(+p)           25.79ng/ul    2.71 uM
 Splint(-p)           31.02ng/ul    3.27 uM
 Probes(+p)           28.47ng/ul    0.62 uM
 Probes(-p)           23.00ng/ul    0.50 uM
  • APV6_Splint (31nt) is 9.5kDa

Test circularization #6[edit]

Amount of template:   (splint to template)     # moles              Concentration
                              2:1            1.25x10^-12         0.042 uM (1.9 ng/ul)
Amount of splint: 2.5x10^-12 moles 

Set up for DMR330K probes:

Reaction using APV6_Splint (+p):
Linear template              30 ul
Ampligase buffer (10x)        5 ul
APV6_Splint (2.71uM)       0.92 ul
Ampligase (5U/ul)             1 ul (add later)
H2O                         9.1 ul
===================================
                             50 ul
Reaction using APV6_Splint (-p):
Linear template              30 ul
Ampligase buffer (10x)        5 ul
APV6_Splint (3.27uM)       0.76 ul
Ampligase (5U/ul)             1 ul (add later)
H2O                         9.2 ul
===================================
                             50 ul
Tests:
S+/P+ no enzyme
S+/P+
S+/P-
S-/P+
S-/P-
Template dilution:
(1) ~Lambda exo digested probes (+p) =  28.47 ng/ul 
     1.9 ng/ul * 95 ul / 28.47 = 6.34 ul probes (+p) + 88.66 H2O
(2) ~Lambda exo digested probes (-p) = 23.00 ng/ul 
     1.9 ng/ul * 65 ul / 23.00 = 5.37 ul probes (-p) + 59.63 H2O
Program:
94 C 3min -> 60C for 10 min (add Ampligase*) ->(96C 30s -> 60C 10 min)x5 -> 94C for 3 min to inactivate enzymes
* I added 1ul ampligase to my no enzyme control so now it S+/P+ is replicate of S+/P+ (no enzyme).
TBU Gel
File:Test Circularization no 6.jpg
A - PNK phosphorylated splint + PNK phosphorylated probes rep #1 (2 ul)
B - PNK phosphorylated splint + PNK phosphorylated probes rep #2 (2 ul)
C - PNK phosphorylated probes + no PNK splint  (2 ul)
D - no PNK phosphorylated probes + PNK phosphorylated splint (2 ul) 
E - no PNK phosphorylated probes + no PNK splint (2 ul)
F - PNK phosphorylated probes (2 ul)
G - no PNK phosphorylated probes (2 ul)
H - PNK phosphorylated splints (2 ul)
I - no PNK splint (2 ul)
J - 10 bp ladder (0.5 ul)

Discussion[edit]

  • Reaction is very inefficient; might be because of old reagents since I don't even see a thick band for the concatamers at the top.