SBB11Ntbk-Anand Kesavaraju

Anand Kesavaraju 08 March 2011 (PST)

Since neither of my parts (sbb1107 & sbb1140) produced viable colonies from the culture tubes, I assisted Jessica Wen with her Analytical Digests (Mapping) protocol:

Procedure

Set up the following 10uL reaction in a PCR tube:

 4uL ddH2O
 4uL Miniprepped plasmid
 1uL 10x NEB Buffer 2
 .5uL EcoRI
 .5uL BamHI (for parts >250bp) or XhoI (for parts <250bp)
 FOR Righty Methylated Parts use Eco/Xho!
 Incubate at 37 on the thermocycler for 30 minutes
 Run an analytical gel
 Take a picture of the gel 

 Calculate the expected fragment sizes
 Are the calculated sizes consistent with the bands on the gel?

User:Anand Kesavaraju 01 March 2011 (PST)

Ligation

Procedure

• Set up the following reaction:
  

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase

• Pound upside down on the bench to mix
  
• Give it a quick spin to send it back to the bottom of the tube
  
• incubate on the benchtop for 30min
  
• Put on ice and proceed to the transformation
  

Transformation

Procedure
1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water to the cells (if greater volume is desired)
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42 (longer incubation may work better)
8. Put back on ice for 1 min
9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

• Plates will be incubated overnight, then colonies will be picked.

User:Anand Kesavaraju 25 February 2011 (PST)

• No class today.

User:Anand Kesavaraju 24 February 2011 (PST)

Zymo Cleanup of Digest

Procedure

Transfer into the Zymo column inside a collection tube (small clear guys)
Spin through, discard waste.
Add 200 uL of A4 Wash Bbuffer (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

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