IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/29

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Team Allergy

 * Ran a gel for our second attempt at amiRNA stiching (stage 1): [[Image:AmiRNA_stich2_stage1.jpg|50px]]. It seems that the backbone is still present at high concentrations after PCR purification. Maybe this is why last time we had a high incidence of background? This time we're gel purifying.


 * Dignostic Digest

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).

Gel Extraction of Barnase insert

 * followed gel extraction protocol in QIAquick Spin Handbook

Nanodrops:

Samples 1-9



 * Lane 1: ladder
 * Lane 2: ladder
 * Lane 3: ACC synthase 1
 * Lane 4: ACC synthase 2
 * Lane 5: Acc synthase 3
 * Lane 6: Terminator 1
 * Lane 7: Terminator 2
 * Lane 8: Terminator 3
 * Lane 9: Act2LacI 1
 * Lane 10: Act2LacI 2
 * Lane 11: Act2LacI 3
 * Lane 12: ladder

insert sizes:


 * ACC synthase: 300 to 350
 * Terminator: unknown
 * Act2LacI: ~1200

Samples 10-18



 * Lane 1: empty
 * Lane 2: ladder
 * Lane 3: Barn1-strep1
 * Lane 4: Barn1-strep2
 * Lane 5: Barn1-strep3
 * Lane 6: Barn2-strep1
 * Lane 7: Barn2-strep2
 * Lane 8: Barn2-strep3
 * Lane 9: EcR2
 * Lane 10: EcR3
 * Lane 11: EcR4
 * Lane 12: ladder

insert sizes
 * Barn1-strep: <400bp
 * Barn2-strep: <400bp
 * Ecr: ~1000bp

Samples 19-24



 * Lane 1: empty
 * Lane 2: ladder
 * Lane 3: empty
 * Lane 4: RxR-Hm 1
 * Lane 5: RxR-Hm 2
 * Lane 6: RxR-Hm 3
 * Lane 7: RxR-Lc 1
 * Lane 8: RxR-Lc 2
 * Lane 9: RxR-Lc 3
 * Lane 10: empty
 * Lane 11: empty
 * Lane 12: ladder

insert size
 * RxR-Lc: 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.

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 10.5μLDH2O

4 tubes of each, 20μL each tube

VP16
 * 10.5μL DH2O
 * 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

Minipreps

 * Miniprep of Mira/Brazz+StrepII+STOP in V0120

Gel Extraction

 * Gel extraction of J45017:


 * 1) Ladder
 * 2) Mira N N/S
 * 3) Mira C N/S
 * 4) Brazz N N/S
 * 5) Brazz C N/S
 * 6) J45004 X/P
 * 7) B21 X/P
 * 8) J45017 undigested
 * 9) Ladder


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