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Team:Stanford/Notebook/Lab Work/Week 9
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8/23 Monday
Chris's Notebook
Miniprep E1 for testing with the plate reader Miniprep of PknH+B0034, Mstp+B0034; sent to sequencing
Inoculations E1, PknH, MstP, EmbR (for freezer stock)
Restriction Digest 4K5 and 4A5 (w/ccdb), I0500, Mstp+B0034, PknH+B0034
Ligation Mstp+B0034: 1600 bp (320 ng) PknH+B0034: 1300 bp (260 ng) EmbR: 1200 bp (260 ng) F2620: 1060 bp (220 ng) I0500: 1200 bp ( 220 ng) J23100+B0034: 100 bp (0.5 ul) pSB4A5: 150 ng (3400 bp) pSB4K5: 150 ng (3400 bp)
I0500+Mstp+B0034 AMP I0500+PknH+B0034 AMP F2620+Mstp+B0034 KAN F2620+PknH+B0034 KAN J23100+B0034+EmbR C
8/24 Tuesday
Chris's Notebook
Sequencing Results: Will use MB3 and PB1; both came back good
Restriction Digests: MB3, PB1; use Ryan's protocol Freezer Stocks E1, PknH, MstP, EmbR Ligations I0500+Mstp+B0034 AMP I0500+PknH+B0034 AMP F2620+Mstp+B0034 KAN F2620+PknH+B0034 KAN J23100+B0034+EmbR C
8/25 Wednesday
Chris's Notebook
Miniprep MB3, I0500 (stimulate with IPTG at 1030; inoculated at 1130 pm last night) Restriction Digest MB3, I0500 (use Ryan's protocol; do in group of two and concentrate) Ligations of Yesterday F2620+PknH+B0034 KAN worked J23100+B0034+EmbR AMP worked I0500+PknH+B0034 AMP no growth
Ligations Today
I0500+PknH+B0034 AMP no growth (use my I0500)3 I0500+Mstp+B0034 AMP F2620+Mstp+B0034 KAN
Inoculate/for Testing E1, MB3
8/26 Thursday
Chris's Notebook
Transformations I0500+PknH+B0034 AMP (Alex's I0500 and Jerome's I0500) Miniprep MB3 Restriction Digest MB3 (X/P) Ligations I0500+Mstp+B0034 AMP F2620+Mstp+B0034 KAN
Inoculate (in at 1230 AM)
3 each from successful transformations (12 total)
8/27 Friday
Chris's Notebook
Ligations I0500+Mstp+B0034 AMP F2620+Mstp+B0034 KAN
Transform (see above)
Restriction Digest Screen
Miniprep: J23100+B0034+EmbR, F2620+B0034+PknH
Restriction Digest of the 12 inoculations (save some for sequencing; just get 500 ng)
Restriction Digest of J64100, psb4A5 (ccdb markers)
Inoculate: I0500+B0034+PknH
