Team:UC Santa Barbara/Notebook

Notebook

7/14/14 - Project began. Started planning project timeline and methods/materials needed to complete project.

7/16/14 - Ordered primers for CysK-T25 OE PCR project, 4 primers total: 
                      
Primer #1: CysK-EcoRI Forward-tttgaattcATGAGTAAGATTTTTGAAGATAACTCG
Primer #2: CysK-OE reverse -  cccgcttgctccagtcccCTGTTGCAATTCTTTCTCAGTG
Primer #3: T25-PstI reverse - tttctgcagaTTATATCGATGGTGCAGCC
Primer #4: T25-OE Forward - gggactggagcaagcgggCAGCAATCGCATCAGGC

7/22/14 John - Began PCR of OE fragments for CysK-T25 
           Enzyme: PFU Polymerase
              PCR Temps: 95 degrees C for 1 minute 
                         50 degrees C for 1 minute 30 seconds
                         72 degrees C 3 minutes 30 seconds

7/23/14 -John + Zack - Digest of CysK-T25 with EcoRI and PstI
                       
                       Protocol: -5uL PCR product, 2uL Buffer 2.1, 12uL H2O, 0.5uL of EcoRI and PstI in an eppendorf tube
                                 -5uL Vector, 2uL buffer 2.1, 12uL H2O, 0.5uL of EcoRI and PstI in an eppendorf tube
                                 -Set both in warm room for 90-120 minutes 

                       After Digest: Run the digest products out on a gel, cut relevant bands out, and purify DNA from gel
                                     Gel Purification Protocol:
                                      *Add 600uL Gel Dissolve to eppendorf tubes containing gel
                                      *Heat tubes in 60 degree waterbath until gel dissolves
                                      *Add 200uL isopropanol once gel dissolves
                                      *Transfer contents of eppendorf tube to gel purification column, let sit for 30 seconds
                                      *Turn on vacuum, then in order add:
                                               500uL PB
                                               750uL PE
                                               250uL PE
                                      *Take gel purification column off vacuum, and spin column in centrifuge at 15K rpm for 2 minutes to collect waste product
                                      *Transfer gel purification column to eppendorf tube, add 50uL elution buffer, spin at 8K rpm for 30 minutes to collect DNA

                      After gel purification, we ran a ligation to ligate the vector and insert together
                                    Ligation Protocol:
                                     *In a microfuge tube, add 16uL vector DNA, 2uL Insert DNA, 2uL 10x ligase buffer, 0.5uL T4 Ligase
                                     *Control Tube: 16uL H2O, 2uL insert DNA, 2uL 10x ligase buffer, 0.5uL T4 Ligase
                                     *Let sit at room temperature for 1 hour or more
 
                     After Ligation: Transform into X90 Cells
                                    Transformation Procedure: 
                                     *Take cells out of -80 freezer, let thaw on ice
                                     *Aliquot 2 eppendorf tubes with 50uL of cells each
                                     *Add 10uL of vector + insert ligation to one and 10uL of H2O + insert ligation to the other 
                                     *Let the eppendorf tubes sit for 15 minutes on ice
                                     *Put eppendorf tubes in 42 degrees celsius water bath for 45 seconds exactly (heat shock)
                                     *After heat shock, add 1mL LB Broth growth medium to tubes using sterile technique and set mixtures in warm room for 1 hour to let cells recover
                                     *Warm up antibiotic-laced agar plate at the same time
                                     *Take cells out, spin at 15K RPM for 2 minutes 
                                     *Remove 950uL of LB media leaving 50uL behind, and then resuspend cells in the leftover 50uL
                                     *Transfer 50uL to agar plate, spread cells, put in warm room overnight.

         7/24/14