SBB12Ntbk-Mike Hwang

Mike Hwang 14:49, 16 February 2012 (EST)

 
Day 1 - Preparation of PCR and PCA

PCA Preparation for Leucine Zipper RLER
Protocol:
1. Added 38uL ddH2O to small PCR tube using p100 micropipette
2. Added 5 uL of 10X expand buffer (labeled with red) using p10
3. Added 5 uL of 2mM dNTPs (labeled with blue) using p10
4. Prepared separate 6uL oligo mixture of 100uM concentration by adding 2 uL each of o1, o2 and o7 oligos and mixed well. To make 100uM solutions of o1 and o2, 922 uL and 961 uL of ddH2O added to stock o1 and o2, respectively. 
5. From above solution, 1 uL was removed and added to the PCR tube
6. Added .75 uL expand polymerase
7. Labeled PCR tube with A, MH and designated as PCA reaction
8. Placed PCR tube on small rack labeled PCA and put on ice


PCR Preparation of cI and oBP constructs
Protocol:
1. Labeled 2 different PCR tubes cI as A and oBP as B. 
2. Added 24uL of ddH2O into both tubes
3. Added 3.3 uL of 10X expand buffer and 3.3 uL of 2mM dNTPs into both tubes
4. Added 1 uL of 10uM Ca998 to tube A
5. Added 1 uL of 10uM ca1333R to tube A
6. Added 1 uL of 10uM 1333F to tube B
7. Added 1 uL of 10 uM g00101 to tube B
8. Added 0.5 uL of jtk2768 to tube A
9. Added 0.5 uL of 9525-1835 to tube B
10. Added 0.5 uL of expand polymerase to both tubes
11. Placed in '500-1000bp' labeled rack on ice

Mike Hwang 16:52, 17 February 2012 (EST)

Day 2 - Zymo Cleanup and Gel Purification of Products

Preparation for pca2 leucine zipper
Protocol:
1. Performed Zymo Cleanup on pca1 product using this protocol ([http://openwetware.org/wiki/Template:SBB-Protocols_Zymo2])
2. Eluted with 51 uL of ddH2O
3. Amplification of Pca1 purified product performed next: added 1 uL of purified pca1 to small PCR tube
4. Added 1 uL of 10 uM o1 oligo
5. Added 1 uL of 10 uM o2 oligo
6. Added 10 uL 5x phusion buffer
7. Added 32.5 uL H2O
8. Added 5 uL of 2 mM dNTPs
9. Added 0.5 uL of phusion and placed on ice prior to assembly

Gel Purification of A+B cI-oBP Preparation
Protocol:
1. Loaded 10 uL A (6 uL of A + 4 uL loading dye) into Gel Lane 6
2. Loaded 10 uL B (6 uL of B + 4 uL loading dye) into Gel Lane 7
3. Cut out bands corresponding to correct fragment size and placed both in eppendorf tube, added 600 uL of ADB Buffer
4. Heated at 55C until gel dissolved
5. Placed in Freezer for storage

Mike Hwang 14:59, 21 February 2012 (EST)

Day 3 - Purification of pca2 and Gel Purification of A+B

Gel Purification of A+B
Protocol:
1. Thawed frozen A+B gel product
2. Transferred contents to Zymo Column inside of collection tube
3. Spun for 15 seconds and discarded waste
4. Added 200 uL Zymo Wash Buffer and spun through (15s), total of two washes
5. Spin for 90 seconds to full dry
6. Eluted with 40 uL of ddH2O
7. Added 0.5 uL of purified A+B to small PCR tube
8. Added 24 uL ddH2O
9. Added 3.3 uL each of 10X expand buffer and 2mM dNTPs
10. Added 1 uL each of 10 uM CA998 and 10uM g00101 oligos 
11. Added 0.5 uL of expand polymerase "1"
12. Placed on ice prior to PCR cloning

Small Fragment Zymo Cleanup for pca2
1. Performed small fragment zymo cleanup protocol ([http://openwetware.org/wiki/Template:SBB-Protocols_Zymo2])
2. Final elution performed with 51 uL of ddH2O

Digestion of pca2 with NheI/BamHI
1. Added 8uL of purified pca2 product
2. Added 1 uL of NEB2 buffer
3. Added 0.5 uL of NheI
4. Added 0.5 uL of BamHI
5. Placed in small PCR tube and then incubated in thermocycler at 37C for 1 hour
6. Added 100 uL ADB buffer to pca2 digest in Eppendorf tube
7. Labeled as 'Digest pca2 not pur.' and placed in freezer box labeled ' 140L eluted products'

Mike Hwang 14:38, 23 February 2012 (EST)

Day 4 - Zymo Cleanup of 1207dig and cI-oBP pcrpdt plus Analytical Gel

Zymo Cleanup of 1207dig
1. Removed tube labeled 'Digest pca2 not pure' from freezer and thawed
2. Added 500 uL of ethanol and transferred to zymo column
3. Performed zymo cleanup with final elution volume of 51 uL using this protocol ([http://openwetware.org/wiki/Template:SBB-Protocols_Zymo2])
4. Froze purified 1207dig

WILL PERFORM LIGATION AND PLATING ON FRIDAY

Analytic Gel of pcrpdt of cI-oBP construct
1. 6 uL of pcrpdt mixed with 4 uL of loading dye and loaded into column 10 of Gel 1
2. Pictures taken of gel fragments to ensure product (A+B) is consistent with the pcrpdt

Zymo Cleanup of cI-oBP construct
1. Performed zymo cleanup on pcrpdt using this protocol ([http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1])
2. Final elution volume of ddH2O was 33 uL
3. Purified pcrpdt, labeled 'MH pcrpdt pure' placed in freezer

WILL PERFORM DIGESTION REACTION FIRST ON THIS PRODUCT FIRST! USING ECORI/BAMHI 

Mike Hwang 17:08, 24 February 2012 (EST)

Day 5 - Digestion of pcrpdt and ligation of 1207dig

Digestion of pcrpdt
Protocol:
1. Prepare digestion reaction for pcrpdt 
2. Add 8uL of pure pcrpdt
3. Add 1 uL of NEB2 buffer
4. Add 0.5 uL each of EcoRI and BamHI (total volume is 10uL)
5. Place on 37C thermocycler for 1 hour
6. Run gel and cut out pcrpdt
7. Melt with 600 uL of ADB buffer and freeze

Ligation and plating of 1207dig
Protocol:
1. Prepare ligation reaction for 1207dig with this protocol
(http://openwetware.org/wiki/Template:SBB-Protocols_Enz4)
2. Mix well and then incubate at benchtop for 30 minutes
3. Placed on ice and proceeded to transformation step.
4. Transformation via this protocol: [PUT HERE]

Mike Hwang 14:04, 28 February 2012 (EST)

Day 6 - Ligation and Plating of 1207dig and PCR prep for 1218
PCR preparation for 1218
Protocol:
1. Added 24 uL of ddH2O to tube A and tube B
2. Added 3.3 uL each of 2mM dNTPs and 10X expand buffer to tube A and tube B
3. Added 1 uL of CA998 and 1 uL of osbb1333R to tube A (10uM)
4. Added 1 uL of osbb1333F and 1 uL of G00101 to tube B (10uM)
5. Added 0.5 uL of jtk2768 to tube A and 0.5 uL of 9525-1835 to tube B
6. Added 0.5 uL of Expand Polymerase to tube A and tube B
7. Placed on ice rack to run PCR program for 500-1000 bp products

Ligation of 1207dig
Protocol:
1. Added 6.5 uL of ddH2O
2. Added 1 uL of T4 Ligase Buffer 
3. Added 1 uL of Bca1834 NheI/BamHI digest 
4. Added 1 uL of 1207dig
5. Added 0.5 uL of T4 DNA Ligase
6. Incubated for 30 minutes on bench after mixing with centrifuge
7. Transformation was performed via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Micro1]

Mike Hwang 15:17, 1 March 2012 (EST)

Day 7 - Gel Purification and Zymo Cleanup of A and B and Miniprepping of 1207 Transformed Bacteria
Purification and PCR Preparation of A+B
1. Loaded 6uL of each A and B into lanes 3 and 4 of agarose gel premixed with 4 uL of loading dye (10 uL total volume loaded)
2. Cut out bands corresponding to ~640 bp in lane 3 and a band corresponding to ~600 bp in lane 4
3. Cut out extra gel matter and then placed into 1.5mL tube with 650uL ADB Buffer
4. Melted gel at 55C and then performed Zymo Gel Purification via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Zymo3]
5. Eluted final volume of 50uL of ddH2O during Zymo Cleanup
6. Prepared PCR reaction using 24 uL of DMSO in PCR tube
7. Added 3.3 uL of 10X Expand buffer and 2mM dNTPs
8. Added 1 uL each of CA998 and G00101 oligos
9. Added 0.5uL of Expand Polymerase '1'
10. Added 0.5uL of pure A+B template DNA
11. Placed on ice for 1k-2k PCR

Mini prepping of 1207 Transformed Bacteria
1. Proceeded with mini prep of transformed bacteria via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Micro3]
2. Purified DNA from both colonies (labeled 1207-1 and 1207-2) were frozen prior to mapping

Mike Hwang 16:13, 2 March 2012 (EST)

Day 8 - Zymo Purification of 1218pcrpdt and Analytical Digest of 1207

Zymo Purification of 1218pcrpdt
Protocol:
1. Performed zymo cleanup via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1]
2. Eluted with 33uL of ddH2O
3. Labeled tube as "Pure 1218 A+B" and side label of "pcrpdt"
4. Freeze until Digestion on 3/6/2012

Analytical Digest of 1207
1. Prepared analytical digest of both 1207-1 and 1207-2 miniprepped plasmids via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Enz6]
2. Used 2uL of miniprep plasmid + 2uL ddH2O instead of 4 uL of plasmid and used NheI and BamHI to digest
2. Fluorescent images of gel bands were taken

Mike Hwang 13:26, 6 March 2012 (EST)

Day 9 - Digestion of 1218pcrpdt and Zymo Cleanup and Sequencing of 1207 mini prep plasmids

Sequencing sbb1207
Protocol:
1. Analyzed gel and visualized expected band sizes in both colonies. Refer to image: [[Image:http://openwetware.org/wiki/Image:2012_03_02_gel3_ssb2012spring.jpg]]
2. 1207-1 is shown in lane 9 and 1207-2 is shown in lane 10
3. The brighter band in each were consistent to ~3000 bp in length, and the faint smaller bands ~100bp in length, as predicted using ApE.
*The 100bp fragment is the LZ_RLER part contained between the NheI and BamHI sites
4. Gave to Chris Anderson for sequencing

Digestion of 1218pcrpdt and Zymo Cleanup
1. Prepared digestion of pure 1218pcrpdt via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Enz2]
2. Ran 1218pcrdig in Lane 8 of Gel 3 (3/6/12) 
3. No bands appeared so SOEING will be done again after gel purification of A and B.
4. SOEing PCR reaction prepared using standard PCR protocol, except using 2.4 uL of DMSO and 21.6 uL of ddH2 to run the reaction
5. Placed on ice for 1k-2k PCR

Mike Hwang 15:10, 8 March 2012 (EST)

Day 10 - Gel Purification of 1218pcrpdt

Zymo cleanup of 1218pcrpdt
Protocol:
1. Performed zymo cleanup via this protocol: [http://openwetware.org/wiki/Template:SBB-Protocols_Zymo1]
2. Eluted with 33uL of ddH2O
3. Labeled tube as "gp1218pcrpdt-MH 3/8"
4. Loaded 4 uL of eluted product with 4uL of dye and performed analytical gel to ensure that product is present before continuing to digestion
5. Small amount of desired product (SOEing A+B) and traces of A and B present
6. Will run digestion on pure1218pcrpdt on 3/13

Mike Hwang 13:13, 13 March 2012 (EDT)

Day 11 - Digestion and Gel purification of 1218 product
1.SOEING failed again...

Protocol for Quantification of homodimerization
Transformation of Reporter Cells
  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 desired plasmid part containing the product to be tested, 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