Jordan

• Ligate YcdO-Cas9 into pSB1C3
• Used a 2:1 molar ratio of insert/backbone
• YcdO-Cas9 with BB ends insert calculated as 3300 bp
• SB1C3 at 2100 bp
• 10 uL reaction volume
• Used "YcdO Cas9, EcoRI SpeI PP 10.3" restriction digest from Paul
• 2 uL (~80 ng)
• Used pSB1C3 EcoRI/SpeI digest PP
• 10.3, 5 uL (25 ng)
• 1.5 uL water
• 1 uL T4 ligase buffer
• 0.5 uL T4 ligase
• Incubating at 25°C for 20 minutes, heat kill at 80°C for 20 minutes
• YcdO concentration in restriction digest was at ~40 ng/ul
• Transform pSB1C3-YcdO-Cas9 into Top10
• Transformed with 3 ul of ligation product
• 30 second heat shock
• 1 hr 15 recovery
• 900uL SOC, plated 450uL
• Plates are titled "YcdO-Cas9 in SB1C3 for sequencing/shipping, TOP10"
• Ligate gRNA template and gRNA guide into pSB1C3
• gRNA guide and gRNA template ratios calculated with 150 bp, SB1C3 calculated with 2100 bp
• Used 2:1 molar ratio of insert:backbone
• 10 uL reaction volume
• "gRNA temp EcoRI SpeI PP 10.3," 2 ul (3.6 ng)
• "gRNA EcoRI SpeI PP 10.3," 2 ul (3.6 ng)
• Restriction digest concentrations were diluted:
• 1:5 for gRNA guide
• Initial: 230 ng in 25 uL reaction=~9 ng/uL to a concentration of 1.8 ng/ul
• 1:50 for gRNA temp
• Initial conc. 2350ng in 25 uL=~90 ng/uL to a concentration of 1.8 ng/ul
• Used 5ul of "pSB1C3 EcoRI SpeI PP 10.3" (25 ng)
• 1.5 ul water
• 1 ul T4 ligase buffer
• 0.5 ul T4 ligase
• Incubating 25°C for 20 minutes, heat kill at 80°C for 20 minutes.
• Ligate gRNA template and gRNA guide into pSB1C3 (again)
• Redid above, but calculated both insert ratios with 1100 bp
• Did not dilute RNA guide, used 3 ul for ~27 ng insert
• Did dilute RNA temp 1:10, but then forgot and used undiluted product.
• Increased reaction volume to 20 ul with water, used 2 ul of buffer
• Added 3 ul or 270 ng (ten times as much insert as needed)
• Transform ligated gRNA products into Top10
• Transformed with 3 ul of ligation product
• 30 second heat shock
• 1 hr 15 recovery
• 900 uL SOC, plated 450 uL
• Plates are titled "BBP-mRFP-gRNA-guide/temp-BBs in SB1C3 for seq/shipping, TOP 10"
• Grow up new overnight cultures (2 of each) for pET28a-Cas9 (BL21) and pET28a-DsbA-Cas9 (BL21) and TorA-Cas9 in pSB1C3 (Top10) and DsbA-Cas9 in pSB1C3 (Top10)
• Grew up new cultures in order to re-miniprep for reasonable concentrations

Paul

• Transform pSB1C3-Cas9 into JC8031
• Transformed 1 uL of "Cas9 plasmid miniprep MC 9.2.16 358.9 ng/uL" (I forgot to dilute the sample oops) into JC8031, and 50 pg of pUC19 [Amp] control into Top10 as transformation control. Recovered in 900 uL SOC, plated 450 uL.
• Restriction digest for YcdO-Cas9 into pSB1C3
• Used "YcdO SD" from PCR purified box 5 ul=~1 ug DNA, digest with 1 uL EcoRI HF and slightly < 1 uL SpeI HF from Kelly
• Restriction digest for gRNA-template & gRNA-guide into pSB1C3
• 10 uL of gRNA 10 JH (23 ng/uL)
• 10 uL of "gRNA template MC 9/2 (235 ng/uL)
• 5 uL of linearized pSB1C3 from iGEM (25 ng/uL)
• *used 1 uL Spei GQ Promega from box because we ran out of SpeI HF from Kelly*

Tasfia

• Miniprep TorA-Cas9 in pSB1C3(Top10) and DsbA-Cas9 in pSB1C3 (Top10)
• Miniprep pET28a-Cas9 (BL21) and pET28a-DsbA-Cas9 (BL21)
• Really low yields