Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/02/08

Current revision

Project name

Type IIS Assembly - Making PCR Products

1A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG

H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA

LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT

LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC

PCR program:

Expected: 1. H2B = 410 2. LOV = 461

Results of the PCR product digests are shown. It was later found that the transparency used was blocking the UV light, so no bands could be seen.

The PCR products were purified with the Zymo DNA Clean & Concentrator Kit and eluted with 20 μL dH₂O

@@ Line 11: / Line 11: @@
 * Final assembly product: pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
 * Primer design
-H2B
+#H2B
 A3/H2B fwd 5'-cacaccaCGTCTCa TAGA ATGCCAGAGCCAGCG
 H2B/LOV rev 5'-cacaccaCGTCTCa CCAA CTTAGCGCTGGTGTA
-LOV
+#LOV
 LOV fwd 5'-cacaccaCGTCTCa TTGGCTACTACACTT
 LOV+his/1A3 rev 5'-cacaccaCGTCTCa TAGT ttagtggtgatggtgatgatgAAGTTCTTTTGCCGC
@@ Line 56: / Line 60: @@
 <center>
-[[Image:Gel_2-8-13.png|Results of the digests are shown.]]
+[[Image:VN_Gel_2-8-13.png|Results of the digests are shown.]]
 [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]]
 </center>
 <center>
-Results of the PCR product digests are shown, with H2B on the left and LOV on the right. The gel was loaded with 5 μL samples.
+Results of the PCR product digests are shown.  It was later found that the transparency used was blocking the UV light, so no bands could be seen.
 </center>
@@ Line 70: / Line 74: @@
 | PCR Product || 260|| 280 || 260/280 || ng/μL
 |-
-| H2B || fill || fill || fill || fill
+| H2B || 0.078|| 0.043|| 1.81 || 77.995
+|-
+| LOV || 0.08 || 0.044 || 1.817 || 80.443
+|-
+| pSB1A3 || 0.023 || 0.014 || 1.727 || 23.386
 |-
-| LOV || fill || fill || fill || fill
 |}