SBB12Ntbk-Karen D. Alvarez

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Karen D. Alvarez 14:43, 16 February 2012 (EST)

==PCR for part sbb1217==
1. ca998/osbb1333R on template pBca9145-jtk2768

a)osbb1333R oligo is 34.6nM, MW=11,944.7, Tm=72.1°C. I prepared a dilute solution of oligos of 10μM.

b) I mixed the following:

 24uL ddH2O
 3.3uL 10x Expand Buffer "2"
 3.3uL dNTPs (2mM in each)
 1uL Oligo ca998, 10uM
 1uL Oligo osbb1333R, 10uM
 0.5uL Expand polymerase "1"
 0.5uL Template DNA

c)Labeled tube

2. osbb1333F/g00101 on template pBca9525-Bca1832

a)osbb1333F oligo is 34.0nM, MW=6,766.4, Tm=60.4°C. I prepared a dilute solution of 10μM.

b) I mixed the following:
 24uL ddH2O
 3.3uL 10x Expand Buffer "2"
 3.3uL dNTPs (2mM in each)
 1uL Oligo g00101, 10uM
 1uL Oligo osbb1333F, 10uM
 0.5uL Expand polymerase "1"
 0.5uL Template DNA

c)Labeled tube

3. Place oligos in ice bucket for PCR program according to predicted PCR product base pair length.

a)ca998/osbb1333R PCR product is 643 bp

b) osbb1333F/g00101 PCR product is 1438 bp

==PCA for sbb1206 part (leucine part)==

1. PCA1 on o1,o6,o2 (pca1)

2.I mixed 1μL of each oligo to a tube

3. I prepared the following mixture:

 38 uL ddH2O
 5 ul 10x expand buffer
 5 ul 2mM dNTPs
 1 ul oligo mixture
 0.75 ul Expand polymerase

4. Labeled tube

5. Place in appropriate ice bucket for PCR

Karen D. Alvarez 23:54, 17 February 2012 (EST)

1. Obtained PCR product A and PCR product B. For each PCR product, loaded 6μL of PCR product with 4μL loading buffer in a single well of a 1% agarose gel. Run gel "gel purify" the products.

2. While gel was running, proceeded to do small-Frag zymo clean up(PCR reaction for fragments smaller than 300bp):

 a. Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction PCA1
 b. Transfer into the Zymo column (small clear guys)
 c. Add 500uL of Ethanol and pipette up and down to mix
 d. spin through (15s), discard waste.
 e. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
 f. spin through (15s), discard waste.
 g. Add 200 uL of Zymo Wash Buffer
 h. spin through, discard waste.
 i. spin for 90 seconds, full speed to dry.
 j. elute with water (spin 60s) into a fresh Eppendorf tube

3. Continue with PCA amplification step using the purified PCA1 product. Prepared the following mixture to obtain PCA2 set up:

 1 μL each outer oligo (10 uM): oligos o1 and o2
 1 μL purified pca product: purified PCA1 from previous step
 0.5 μL phusion
 10 μL 5x phusion buffer
 5 μL 2mM dNTPs
 32.5 μL ddH2O

4. Perform PCR on PCA2 solution with the following program:

 a. 2 min initial denature at 94°C
 b. 30 sec denature at 94°C
 c. 30 sec anneal at 60°C [This should be high, as your outer oligos now have a huge overlap with the correct product]
 d. 30 sec extension at 68°C
 e. repeat 2-4 30 times total

5. After gel finished running bands were observed using UV light. Bands roughly correspond to their respective sizes (PCR product A=643 bp and B=1438 bp)

 a. Cut the bands and place in a tube
 b. Add about 650 μL of ADB buffer
 c. Place tube in water bath to melt the agarose
 d. Put away in freezer for further manipulations in NEXT lab class.