Team Allergy
- amiRNA fragment production appears to have worked at every Tm we tried (click images for details):
- V9/V10
- Diagnostic digest of our gel :
[[Image: |240 px]]
(Should see ~ 7kb band and an 840 band)
- Realized that we hadn't gel purified our inserts
- Gel purification of inserts (entire ihpRNA parts)
[[Image: |240px|Gel 2]]
Gel 1: Ladder, 9, 11c1, 11c2, ladder, 25c1, 28c1, 28c2, 36c1, 36c2, ladder
Gel 2: Ladder, 37c1, 37 c2, 38c1, 38c2, ladder
Team Fence
PCR of VP16.3
60μL reaction:
- 1.5 μL pfx polymerase
- 3μL DNTPs
- 18μL enhancer buffer
- 1.5μL MgSO4
- 3μL 1/10 dilution VP16.Hind.Fwd
- 3μL 1/10 dilution VP16.Rev
- 3μL 1/10 dilution of VP16 (1)
- 12μL amp buffer
- 5μL DH2O
Using program:
1=94°C for 2:00
2=94°C for :15
3=60/68°C for :30
4=68°C for :10
5=Goto 2, 30 times
6=4.0°C forever
7=end
Digestion of Gal-EcR
BamH1 and Nco1
12 digestions, one for each Gal-EcR miniprep
Each:
- 1μL BamH1
- 1μL Nco1
- 1μL 10x FD green buffer
- 2-7μL EcR-Gal miniprep
- 5-0μL DH2O, to bring total volume to 10μL
Ran a 1% gel of the digest
Lane
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9
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14
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EcR Digest 1
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EcR Digest 2
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EcR Digest 3
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EcR Digest 5
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EcR Digest 6
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EcR Digest 7
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EcR Digest 8
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EcR Digest 9
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EcR Digest 10
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EcR Digest 11
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EcR Digest 12
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KB+ ladder
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Team Flavor
Confirmation Digests
- Ran confirmation digests of miniprepped J45004-1, J45004-2, J45017-1, J45017-2
- Digested approximately 500ng with xbaI/speI
- Ran confirmation digests of miniprepped Miraculin N strep+stop and V24, Miraculin C strep+stop and V24, Brazzein N strep+stop and V24, Brazzein C strep+stop and V24.
- Digested 400-500ng with notI/speI
Retry Ligating j45004 and v0120
- Used PCR purified J45004 that had been previously digested with xbaI/pstI
- Used v0120 from Team Vector that they had digested with xbaI/pstI and used in a successful ligation
- 3:1 insert to backbone ration used in ligation
- 52ng of insert and 50ng v0120 backbone
- J45004 insert is 1100 bp
- Transformed and plated on LB+Amp plates and left overnight
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