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.