Sbb14-GJordanRay
Graham Jordan Ray 15:31, 15 April 2014 (EDT)
Digested minipreps with ncoI and hindIII
Messed up gels. Oh well.
Repeating.
Graham Jordan Ray 14:38, 10 April 2014 (EDT)
miniprepped all 44 liquid cultures
next time we will map and give Chris good samples for sequencing
Graham Jordan Ray 14:45, 8 April 2014 (EDT)
pick 4 colonies from each plate in to media with amp in it
Counts of cells:
Gly: 90
His: 100
Lys: 100
Glu1: 70
Glu2: 60
Ile: 40
Ala: 40
Asp: 35
Cys: 30
Phe: 50
Leu: 70
forgot to do control
Graham Jordan Ray 13:02, 3 April 2014 (EDT)
Today we ligated all 11 of our digests with digested DNA.
After we transformed into cells and plated onto Amp plates
Graham Jordan Ray 12:46, 1 April 2014 (EDT)
Digested more template on 3/20:
Graham Jordan Ray 15:13, 18 March 2014 (EDT)
Ran a digest on all samples according to map below and ran a gel.
lane 1:ladder, lane2:glu2 (.6&.8 parts), lane3:Ile(2.8) lane 4: leu (2.5) lane 5: vector (4) lane 6: Cys (1.4) lane 7: Asp (1.8) lane 8: Glu (1.5) lane 9 phe (3.4) lane 10: Gly (2.9)
lane 1:ladder, lane2:his (1.3), lane3: Lys(1.5) lane 4: Ala (2.6) lane 5: vector (4 wrong no enzyme) lane 6: vector (4 wrong no enzyme) lane 7: empty lane 8-10:other group
Graham Jordan Ray 13:43, 13 March 2014 (EDT)
Ran a gel on Ala SOEing PCR- added more of tempates this time (5 uL of each)
lane 1: ladder, lane 2: ala, lane 3: other groups sample
Zymo'd Ala sample
Next tuesday we will digest, and gel purify. If time we can ligate and transform!
Graham Jordan Ray 13:09, 11 March 2014 (EDT)
Ran a gel on 2 SOEing PCRs and 2 re-trys of Glu PCRs ( both at 2k45 one with and one without DMSO)
lane 1: ladder, lane 2: ala, lane 3: leu, lane 4: glu(1) +DMSO, lane 5: glu(1) normal
Did Zymos on 3 parts that worked. Now have 10/11 of our parts
Reran SOEing PCR on Ala
Graham Jordan Ray 15:14, 7 March 2014 (EST)
Plan for today:
Look at images of gels all looked good except glu1
Repeating Glu1, failed 1st time
Set up SOEing PCRs.
Digest vector and parts, run on gel, and then do a gel purification on tuesday
NcoI/HindIII (Buffer 2):
- Vector
- Cys
- Asp
- Glu
- Phe
- Gly
- His
- Lys
- Ala
BsaI/HindIII (Buffer 3):
- Ile
- Leu
BsmbI/HindIII (Buffer 3):
- Glu(2)
Graham Jordan Ray 13:10, 6 March 2014 (EST)
Plan for today:
Run gel on pre-soeing PCRs (ALA and LEU) & make sure they are the correct size.
If they are the correct sizes gel purify, and run SOEing PCR.
Concurrently for simple PCRs we are running a gel to check sizes and then we will zymo PCRs to cleanup.
Results of todays Gels:
All 4 SOEing parts look good and right sizes:
lane 1: ala1, lane 2: ala2, lane 3: leu1, lane 4: leu2, lane 5: ladder
All but 1 of normal PCRs look good:
lane 1: ladder, lane 2: cys (1.4), lane 3: asp (1.7) lane 4: glu (1.5) lane 5: phe (3.4) lane 6: gly (2.9) lane 7: his (1.2) lane 8: ile (2.8) lane 9 lys (1.5) lane 10:Glu(2) (1.4)
Graham Jordan Ray 12:56, 4 March 2014 (EST)
Today we started assembly of our parts.
We PCRd on all of our parts off of the genome based on the design file
Graham Jordan Ray 13:16, 18 February 2014 (EST)
our design spreadsheet: Group 3 desing
Graham Jordan Ray 13:16, 13 February 2014 (EST)
On Team 3:
Our amino acids are A,C,D,E,F,G,H,I,K,L
Our organism is Thermotoga maritima.
Found at ATCC Link
Genomic DNA found at Thermotoga maratima