SBB12Ntbk-Mike Hwang

[[User: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

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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