Editing
Brandon:LabNotes/Project1/2012-10-26
(section)
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!
===Results=== '''Nextera results and gels''' *nanodrop results for nextera samples {| {{table}} | align="center" style="background:#f0f0f0;"|'''#''' | align="center" style="background:#f0f0f0;"|'''sample''' | align="center" style="background:#f0f0f0;"|'''DNA''' | align="center" style="background:#f0f0f0;"|'''''' |- | 7|| 150K cells nextera tagmentation||111.85||ng/ul |- | 8|| 50K cells nextera tagmentation||165.6||ng/ul |- | 9|| 10K cells nextera tagmentation||169.59||ng/ul |- | 10|| 1000 cells nextera tagmentation||117.73||ng/ul |- | 11|| 500 cells nextera tagmentation||94.03||ng/ul |- | 12|| 100 cells nextera tagmentation||57.09||ng/ul |- | 13|| 60 ng purified DNA nextera tagmentation||128.13||ng/ul |- | 14|| 6 ng purified DNA nextera tagmentation||101.87||ng/ul |- | 15|| 3 ng purified DNA nextera tagmentation||85.11||ng/ul |- | 16|| 600 pg purified DNA nextera tagmentation||93.49||ng/ul |- | 20|| 1000 cells lysed without tspsome nextera tagmentation||11.68||ng/ul |- | 21|| pure DNA only (6 ng) nextera tagmentation||14.33||ng/ul |- | 22|| Nuclease free H20 only nextera tagmentation||23.32||ng/ul |- | |} *ran gel after 2 step AMPure beads purification (addition of illuminia primers, barcodes) [[File:ZhangLab 2 2012-10-31 15hr 50min-labeled.jpg|600px]] [[File:ZhangLab 2 2012-10-31 15hr 54min-labeled.jpg|600px]] *qPCR curves for blue/orange amplification of products *all products PCR amplification curves *samples in order Samples: 7. 150K cells nextera tagmentation 8. 50K cells nextera tagmentation 9. 10K cells nextera tagmentation 10. 1000 cells nextera tagmentation 11. 500 cells nextera tagmentation 12. 100 cells nextera tagmentation 13. 60 ng purified DNA nextera tagmentation 14. 6 ng purified DNA nextera tagmentation 15. 3 ng purified DNA nextera tagmentation 16. 600 pg purified DNA nextera tagmentation 20. 1000 cells lysed without transposome complex nextera tagmentation 21. pure DNA only (6 ng) nextera tagmentation 22. Nuclease free H20 only nextera tagmentation [[File:2012-10-30 round 3 accessibility nextera orange blue amplification.bmp|600px]] *only nextera treated accessibility samples, amplification curves *samples in order Samples: 7. 150K cells nextera tagmentation 8. 50K cells nextera tagmentation 9. 10K cells nextera tagmentation 10. 1000 cells nextera tagmentation 11. 500 cells nextera tagmentation 12. 100 cells nextera tagmentation [[File:2012-10-30 round 3 accessibility nextera orange blue amplification cells acc.bmp|600px]] *only nextera treated pure DNA samples and controls, amplification curves *samples in order Samples: 13. 60 ng purified DNA nextera tagmentation 14. 6 ng purified DNA nextera tagmentation 15. 3 ng purified DNA nextera tagmentation 16. 600 pg purified DNA nextera tagmentation 20. 1000 cells lysed without transposome complex nextera tagmentation 21. pure DNA only (6 ng) nextera tagmentation 22. Nuclease free H20 only nextera tagmentation [[File:2012-10-30 round 3 accessibility nextera orange blue amplification pure dna, controls.bmp|600px]] '''IVT amplification results/gels''' *Used 1 uL for TBU gel, 1 uL for Qubit quantitation of RNA. RNA eluted in 14 uL N-H20 total. {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''[sample]''' | align="center" style="background:#f0f0f0;"|'''total ng''' |- | 1. 1000 cells IVT||20 ng/ul||280 ng |- | 2. 500 cells IVT||25.1 ng/ul||351.4 ng |- | 3. 100 cells IVT||37.3 ng/ul||522.2 ng |- | 4. 6 ng purified DNA IVT||61.5 ng/ul||861 ng |- | 5. 3 ng purified DNA IVT||60.9 ng/ul||852.6 ng |- | 6. 600 pg purified DNA IVT||60.4 ng/ul||845.6 ng |- | 17. 1000 cells lysed w/out transposome complex IVT||Out of Range||Out of Range |- | 18. pure DNA only (6 ng) IVT||Out of Range||Out of Range |- | 19. Nuclease free H20 only IVT||Out of Range||Out of Range |- | |} *graph of ng RNA versus cell number for IVT RNA amplification. for accessibility samples gave opposite of expected results, not sure why. possible containment? dunno. regardless still gave satisfactory amounts of RNA from IVT amplification, slightly less then accessibility round2. possible that freezer (-20) defreeze had an effect. [[File:Picture1-round3-qubit.png|600px]] *RNA after IVT amplification, TBU gel. Expected more RNA in accessibility samples but still satisfactory total amounts. [[File:ZhangLab 2 2012-10-31 17hr 11min-labeled.jpg|600px]] *qPCR curves for barcode/adaptor addition *not sure what happened. diluted SYBR green from 10,000X to 250X and added to KAPA robust PCR mix, which didn't have SYBR green initially. Where first reading started out is what is expected based on cell number/DNA amounts in the samples. However ran gel and looked fine Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT [[File:2012-10-30 round 3 accessibility IVT barcodes.bmp|600px]] *TBE gel after barcode/illuminia adaptor addition. TBE gel, 50 uL sample total fragments above 127 bp will have an insert, at least 157 for good insert 5β end GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 3β end T20VN_PE_R AAAAAAAAAAAAAAAAAAAAACTAGCCTTCTCGCCAAGTCGTCCTTACG 3β end of PCR_R.N2Ind[XX] with ILA adaptor GCTCGAACATTAGAGCATACGGCAGAAGACGAAC [[File:ZhangLab 2 2012-11-01 16hr 43min-labeled.jpg|600px]] *TBU gel after barcode/illuminia adaptor addition. TBE gel had thick bands so wanted to make sure it was still right sized fragments and not DNA hairballs. [[File:ZhangLab 2 2012-11-01 18hr 02min-labeled.jpg|600px]] *gel size selection [[File:ZhangLab 2 2012-11-09 11hr 33min.jpg|600px]] *validating gel size selection [[File:ZhangLab 2 2012-11-09 15hr 32min-labeled.jpg|600px]] *cloning and transformation results *shown to be uninformative. samples that were successful in last run had bands between 400-600 after PCR amplification. was expected this was not working based on previous gels and barcodes addition results *has newly annealed trasposome in lane 10 only to check. [[File:ZhangLab 2 2012-11-13 15hr 15min-labeled.jpg|600px]] [[File:ZhangLab 2 2012-11-13 15hr 33min-labeled.jpg|600px]] [[File:ZhangLab 2 2012-11-13 15hr 34min-labeled.jpg|600px]] '''conclusions''' *seems like something went wrong since all the curves went down during PCR and the gels did not look corrected. Expected to see different amounts based on input DNA. *IVT samples did not amplify as much as [[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-8-6 round2 IVT data]]. when comparing TBU gel smears they amplified alot more in round2. Possible reasons are the -20 defreeze over the weekend (since someone left the door open) could have left the IVT kits and taq polymerases etc thawed for more than a day. Could also be cell lysis did not occur as efficiently. **ordered new IVT kit and taq polymerases. will remake cell lysis buffer when performing experiment again. *kun mentioned Rui got the same type of smears before and did cloning and transformation on them, and they did not turn out to well. Probably the same issue here *Nextera samples worked fine and gave expected results. Will keep them for next sequencing run for when IVT samples work correctly. *cloning and transformation uninformative. showed that didnt work.
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