IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/29: Difference between revisions
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===Digest of Wednesday's minipreps=== | ===Digest of Wednesday's minipreps=== | ||
===Gel Extraction of Barnase insert=== | |||
*followed gel extraction protocol in QIAquick Spin Handbook | |||
Nanodrops: | |||
{| border="3" style="margin-left: 3em;" | |||
|- | |||
! Plasmid !! Quantity (ng/μL) !! 260/280 | |||
|- | |||
! Tube #1 | |||
| 9.7 || 1.79 | |||
|- | |||
! Tube #2 | |||
| 17.4 || 1.57 | |||
|- | |||
! Tube #3 | |||
| 2.7 || 1.24 | |||
|- | |||
|} | |||
====Samples 1-9==== | ====Samples 1-9==== | ||
Line 79: | Line 100: | ||
*Lane 7: Barn2-strep2 | *Lane 7: Barn2-strep2 | ||
*Lane 8: Barn2-strep3 | *Lane 8: Barn2-strep3 | ||
*Lane 9: | *Lane 9: EcR2 | ||
*Lane 10: | *Lane 10: EcR3 | ||
*Lane 11: | *Lane 11: EcR4 | ||
*Lane 12: ladder | *Lane 12: ladder | ||
Line 109: | Line 130: | ||
*RxR-Lc: 600-700bp | *RxR-Lc: 600-700bp | ||
*RxR-Hm: 600-700bp | *RxR-Hm: 600-700bp | ||
====Summary==== | |||
*We see one appropriately sized band (~350bp) from the ACC synthase miniprep #1, the others did not have any bands. Therefore henceforth we will only use plasmid from miniprep #1 for our reactions. | |||
*Terminator size unknown at present. | |||
*For the Act2LacI promoter we saw in all three lanes the insert sized at ~1200bp. All three minipreps should be fine to use for future reactions. | |||
*The Barnase-strep lanes all displayed inserts of the correct size, ~350 to 400 bp. All minipreps should be good for future reactions. | |||
*The Ect. Receptor insert appears to be about the right size, >1000bp, although the ladder is a bit hard to decipher, all minipreps should be good for future reactions. | |||
*The RxR-Hm did not show any evidence of the presence of an insert. | |||
*The RxR-Lc did show evidence of the presence of an insert, although it may be a bit larger than the expected size (600-700bp). Blurriness of the ladder makes it difficult to decipher. | |||
===Nanodrop of Gal4DBD Minipreps (from the beginning of the summer=== | |||
{| border="3" style="margin-left: 3em;" | |||
|- | |||
! Plasmid !! Quantity (ng/μL) !! 260/280 | |||
|- | |||
! Gal4DBD #1 | |||
| 26.5 || 1.89 | |||
|- | |||
! Gal4DBD #3 | |||
| 16.7 || 1.85 | |||
|- | |||
! Gal4DBD #4 | |||
| 19.1 || 1.83 | |||
|- | |||
! Gal4DBD #5 | |||
| 82.2 || 2.16 | |||
|- | |||
|} | |||
===PCR of VP16 and Gal4DBD (preparing for yeast vector insertion)=== | |||
Gal4DBD | |||
*2μL DNTPs | |||
*.5μL phusion polymerase | |||
*4μL 5x phusion buffer | |||
*1μL 1/10 dilution of BB.Gal4DBD.Rev | |||
*1μL 1/10 dilution of Bam.Gal4.Fwd | |||
*1μL 1/2 dilution Gal4DBD #3 (made 1/2 dilution such that 1μL will be <10 ng/μL | |||
*μL 10.5μLDH<sub>2</sub>O | |||
4 tubes of each, 20μL each tube | |||
VP16 | |||
*10.5μL DH<sub>2</sub>O | |||
*2μL DNTPs | |||
*.5μL phusion polymerase | |||
*4μL 5xphusion buffer | |||
*1μL 1/10 dilution of VP16.Hind.Fwd | |||
*1μL 1/10 dilution of VP16.Rev | |||
*1μL 1/10 dilution of VP16 #1 | |||
PCR program: | |||
VP16 Gal4 | |||
1= 98°C for 10:00 | |||
2= 98°C for :15 | |||
3= 50°C to 60°C for :30 | |||
4= 72°C for 1:30 | |||
5= goto 2, 29 times | |||
6= 72°C for 10:10 | |||
7= 4°C forever | |||
==Team Flavor== | ==Team Flavor== | ||
Line 114: | Line 205: | ||
*Miniprep of Mira/Brazz+StrepII+STOP in V0120 | *Miniprep of Mira/Brazz+StrepII+STOP in V0120 | ||
===Gel Extraction=== | ===Gel Extraction=== | ||
*Gel extraction of J45017: | *Gel extraction of J45017: | ||
[[Image:Mira_brass_ss_digest.jpg||240px]] | |||
# Ladder | |||
# Mira N N/S | |||
# Mira C N/S | |||
# Brazz N N/S | |||
# Brazz C N/S | |||
# J45004 X/P | |||
# B21 X/P | |||
# J45017 undigested | |||
# Ladder | |||
Revision as of 06:54, 3 August 2010
iGEM iGarden | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||
Team Allergy
Gel: ladder, 9, 9, 11, 11, 25, 25, 28, 28, ladder, 36, 36, 37, 37, 38, 38, ladder, LTPS+ PDK, LTPS+PDK, Bet2S+PDK, Bet2S+PDK, GerS+PDK, GerS+PDK, GerS+PDK, GerS+PDK Expected band lengths: Complete Parts 9,11 (LTPS+Pal+LTPA): 840bp and 3200 bp 25,28 (Bet2S+Pal+Bet2A): 840bp and 3200 bp 36,37,38 (GerS+Pal+GerA): 840bp and 3200 bp Sense + Intron Parts LTPS+ PDK: 1200 and 3200 bp Bet2S+ PDK: 1200 and 3200 bp GerS+PDK: 1200 and 3200 bp (9 c1, 25 c2, 36 c1 have a 540 and 3200 band as opposed to a 840 and 3200 band--prbly b/c sense+intron is there but antisense was not ligated in). All intron+sense parts except for LTPS+PDK appear to have 300 bands as well indicating that PDK was not ligated in. (a small mix up with two of the lids was made and its possible that LTPS+PDK (w/ an 840 and 3200 band is actually the complete Bet2 part so we're sending this in for sequening anyway). Team FenceDigest of Wednesday's miniprepsGel Extraction of Barnase insert
Nanodrops:
Samples 1-9
insert sizes:
Samples 10-18
insert sizes
Samples 19-24
insert size
Summary
Nanodrop of Gal4DBD Minipreps (from the beginning of the summer
PCR of VP16 and Gal4DBD (preparing for yeast vector insertion)Gal4DBD
4 tubes of each, 20μL each tube
1= 98°C for 10:00 2= 98°C for :15 3= 50°C to 60°C for :30 4= 72°C for 1:30 5= goto 2, 29 times 6= 72°C for 10:10 7= 4°C forever Team FlavorMinipreps
Gel Extraction
|