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