SBB11Ntbk-Anand Kesavaraju

User:Anand Kesavaraju 25 February 2011 (PST)

• No class today.

User:Anand Kesavaraju 24 February 2011 (PST)

• Zymo Gel Purification

User:Anand Kesavaraju 22 February 2011 (PST)

• Eco/Bam digest PCR products protocol today:

• 8uL PCR material
• 1uL NEB Buffer 2
• 0.5 uL EcoRI
• 0.5 uL BamHI

When you do this, you'll run those digests in the thermocycler, so use PCR tubes to set them up. Also, you should NOT add DNA loading buffer before sending your samples upstairs to be run on the gel. It is dangerous to let an active digestion reaction sit with the extra glycerol present in the loading buffer--it can cause degradation of your DNA. You add the loading buffer right before loading the DNA into the gel.

• Extract DNA by cutting out bands corresponding to sbb1107 and sbb1140 lanes in gel (lane 4, lane 5 respectively)

• Melt in 600 uL ADB buffer at 55 degrees C.

User:Anand Kesavaraju 21 February 2011 (PST)

• Received email from Professor Anderson:

"I noticed on Friday that many of you were using the "AW Wash Buffer" for your Zymo cleanups rather than the "A4 Wash Buffer". Unfortunately, they are both called "Wash Buffer" but they are not the same. AW is equivalent to the Qiagen PB Buffer--it has quanidinium chloride in it. What you wanted was A4, which is ethanol/water. So, your DNA is probably now in the landfill.

I redid all your PCRs and Zymo cleanups and threw out all the old tubes. So, you're back on schedule. I've also modified the protocols and have ordered Qiagen minipreps to eliminate the confusion around the two kits. If you are printing out the protocols, please reprint them before doing things. These are the protocols you will be using:

• http://openwetware.org/wiki/Template:SBB-Protocols_Micro3
• http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1
• http://openwetware.org/wiki/Template:SBB-Protocols_Zymo3

Gel Images:

sbb1140 - lane 7: 3985bp File:Sbb1140AnalyticalGel1.jpg

sbb1107 - lane 3: 1215bp File:Sbb1107AnalyticalGel4.jpg

User:Anand Kesavaraju 18 February 2011 (PST)

• Analytical Gel for sbb1140 - see Figure 2 below. No band appeared in lane 5, indicating PCR was unsuccessful..

User:Anand Kesavaraju 17 February 2011 (PST)

• Repeat sbb1140 (BseRImet) PCR - did not work last time.

• Zymo Cleanup (not small fragment) Protocol:

• Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
• Transfer into the Zymo column (small clear guys)
• Spin through, discard waste.
• Add 200 uL of A4 Wash Buffer (which is basically 70% ethanol)
• Spin through, discard waste.
• Add 200 uL of A4 Wash Buffer
• Spin through, discard waste.
• Spin for 90 seconds, full speed to dry.
• Elute with water into a fresh Eppendorf tube, use the same volume of water (8μL) as the volume of the original reaction

• Run analytical gel for both (just P_malP for today - see Image below, lane 7)

User:Anand Kesavaraju 15 February 2011 (PST)

PCR of sbb1140 and sbb1107

• Only PCR today
• Oligos sbb1140F/R are freeze dried, so rehydrate with appropriate volume of ddH2O and perform a 1:10 dilution to get 10 uM
• sbb1107F/R are already at 10 uM

• Expand buffer/polymerase for genomic (MG1655 DNA)
• Phusion buffer/polymerase for plasmid DNA (pBjh1601CA-Bgl009)

Followed the following Protocol for Cloning by PCR.

User:Anand Kesavaraju 11 February 2011 (PST)

I herd u liek notebooks, so I put a notebook in ur notebook so you can look at Anand's Personal Page