Editing
Dinh 2011/NOTES/2011-9-21
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
==DMR330K E-mix with 5/Phos/ primers== *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== *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== 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== *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.
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information