PLenti-IRES-TFR-EGFP

CloneID: 252

Date: 2011-6-24to 2011-6-28

PCR, DIGESTION, GEL ELECTROPHORESIS:

PCR1) 1ul F1, 1ul P8, 2ul primer mix, 5ul 5x buffer, 0.5ul 50x dNTP, 14.5ul H2O, 1ul pfu enzyme

PCR2) 1ul F2, 1ulR, 1ul EGFP template, 5ul 5x buffer, 0.5ul dNTP, 15.5ul H2O, 1ul pfu enzyme

PCR3) 5 ul PCR1, 5ul PCR2, 1ul F1, 1ul R, 8ul 5x buffer, 1ul dNTP, 2ul pfu enzyme, 27ul H2O

Used MluI+XhoI enzymes to digest, 100% in buffer 3

1) pLentiplus vector: 5ul pLentiplus, 0.4ul Xho1, 0.4ul Mlu1, 1.5ul buffer 3, 7.7ul H2O

2) PCR3 product: 12.7ul purified DNA, 0.4ul Xho1, 0.4ul Mlu1, 1.5ul buffer 3 (75ºC for ~15 min after digestion complete)

Ran agarose gel for pLentiplus vector and PCR3 product

(Image to be uploaded later)

Lanes:

COLONY SCREENING:

PCR:

11 colonies -

each contains: 1ul template (H2O+colony), 1ul A3, 1ul B12, 2.5ul 10x buffer, 0.8ul Taq enzyme, 0.2ul dNTP, 18.5ul H2O

Lanes:

Marker

1: Marker

2: colony 1

3: colony 2

4: colony 3

5: colony 4

6: colony 5

7: colony 6

8: colony 7

9: colony 8

10: colony 9

11: colony 10

12: colony 11

Took DNA from colonies 5-8 and 10-11

(pg. 001143)