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) ***Attached below***

 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.

Karen D. Alvarez 14:45, 21 February 2012 (EST)

1. Continued with SOEing reaction of part ssb1217. I did Zymo Gel Purification procedure.

 a. Obtain Eppendorf tube containing the two gel fragments with buffer.
 b. Transfer into the Zymo column inside a collection tube (small clear guys)
 c. Spin 15 seconds, discard waste.
 d. Add 200 uL of Zymo Wash Bbuffer (which is basically 70% ethanol)
 e. Spin 15 seconds, discard waste.
 f. Add 200 uL of Zymo Wash Buffer
 g. spin through, discard waste.
 h. spin for 90 seconds, full speed to dry.
 i. Elute with 50 μL water (supposed to be 33 μL ) into a fresh Eppendorf tube

2. Set up the second PCR reaction for the purified eluded solution above:

 24uL ddH2O
 3.3uL 10x Expand Buffer "2"
 3.3uL dNTPs (2mM in each)
 1uL Oligo ca998, 10uM
 1uL Oligo g00101, 10uM
 0.5uL Expand polymerase "1"
 0.5uL Template DNA (eluded solution)

a. Run PCR program as follows:
 2 distinct cycling phases—the first 10 cycles have shorter extensions than the latter 20 cycles. You choose which one you  
 want based on the length of your predicted product. If it is under 2kb, use 2K55. If it is between 2kb and 4kb, use 4K55. If 
 it is over 4kb, use 8K55. In each case, start with the "55" version of the program. If you get no product or poor yield of 
 product, repeat the same PCR reaction as two different variants, both with the “45” program. The first reaction will be the 
 same composition as the first. For the second one, don’t add 3.3 uL of the water and instead use the volume for 3.3 uL of  
 DMSO (dimethylsulfoxide)

3.Perform Small-Frag Zymo Cleanup on PCA2 product (leucine zipper part sbb1206 ~142 bp)
 (procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction for 
 fragments smaller than 300bp. It also will remove the buffer and restriction enzymes from a restriction digest
 reaction)

 Add 100 uL of Zymo ADB buffer (brown bottle) to the reaction
 Transfer into the Zymo column (small clear guys)
 Add 500uL of Ethanol and pipette up and down to mix
 spin through (15s), discard waste.
 Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
 spin through (15s), discard waste.
 Add 200 uL of Zymo Wash Buffer
 spin through, discard waste.
 spin for 90 seconds, full speed to dry.
 elute with 50 μL water (spin 60s) into a fresh Eppendorf tube

4. Follow Digestion eluded PCA2 product NheI and BamHI as follows:

 8μL of eluted PCA2 product
 1μL of NEB Buffer 2
 0.5 μL NheI
 0.5 μL BamHI
 
a. Incubate at 37°C on the thermocycler for 1hr
b. Add this PCA2 to 100μL ADB buffer.
c.Put away for NEXT lab

Karen D. Alvarez 15:00, 23 February 2012 (EST)

1. Obtain PCA2 digest product containing ADB buffer (from last lab class). Do small fragment zymo clean up as follows:
 a.Transfer into the Zymo column (small clear guys)
 b.Add 500uL of Ethanol and pipette up and down to mix
 c.spin through (15s), discard waste.
 d.Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
 e.spin through (15s), discard waste.
 f.Add 200 uL of Zymo Wash Buffer
 g.spin through(15s), discard waste.
 h.spin for 90 seconds, full speed to dry.
 i.elute with 8μL ddwater (spin 60s) into a fresh Eppendorf tube

2. Do Regular Zymo Clean up on PCR product (A+B) as follows:
 a.Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
 b.Transfer into the Zymo column (small clear guys)
 c.spin through, discard waste.
 d.Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
 e.spin through, discard waste.
 f.Add 200 uL of Zymo Wash Buffer
 g.spin through, discard waste.
 h.spin for 90 seconds, full speed to dry.
 e.elute with 33 μL water into a fresh Eppendorf tube 

3.Digest "prcprd" on sbb1217
 8uL of eluted PCR product
 1uL of NEB Buffer 2
 0.5uL EcoRI
 0.5uL BamHI

 a. Incubate at 37°C on the thermocycler for 1hr ( did 50 minutes)
 b. Run an agarose gel, and melt with 600μL ADB buffer at 55°C. 

4. Perform Ligation reaction on PCA2 cleaned product (REDO this NEXT class)